In Vivo Bioassay of Recombinant Human Growth Hormone Synthesized in B.mori Pupae

【基金项目】国家自然科学基金（81974347）；中国博士后基金（2021M702351）【作者单位】四川大学华西医院骨科，成都610041【作者简介】吴元刚（1989-），男，四川省成都人，博士，讲师、主治医师，研究方向：骨质疏松和骨关节炎【通信作者】沈彬（1971-），男，四川省成都人，博士，教授、主任医师，研究方向：骨质疏松和骨关节炎，E-mail：********************∙基础研究∙吴元刚,曾羿,李明阳,等.重组Tet-on COLIA1基因腺病毒骨组织靶向表达对大鼠骨质疏松骨折愈合生物力学影响的实验研究[J].中华骨与关节外科杂志,2022,15(3):170-175.重组Tet-on COLIA1基因腺病毒骨组织靶向表达对大鼠骨质疏松骨折愈合生物力学影响的实验研究吴元刚，曾羿，李明阳，刘渊，裴福兴，沈彬【摘要】目的：研究重组Tet-on COLIA1基因腺病毒骨组织靶向表达对大鼠骨质疏松骨折愈合生物力学的影响。

方法：40只健康雌性大鼠按照随机数字表法分为手术组（n=36）和假手术组（n=4）。

手术组大鼠切除双侧卵巢，建立骨质疏松动物模型。

假手术组大鼠仅切开皮肤，不切除双侧卵巢。

手术组36只大鼠切除双侧卵巢3个月后，随机选取4只并处死，同时处死假手术组4只大鼠。

收集其胫骨标本，采用双能X线测量假手术组和手术组大鼠的骨密度，以证实大鼠骨质疏松模型建立成功。

随后，将剩余的32只手术组大鼠，采用薄电锯片从胫骨中段横行切断骨干，克氏针固定骨折断端，以构建大鼠骨质疏松骨折模型。

同时按照随机数字表法分为4组：A组，重组Tet-on COLIA1基因腺病毒+强力霉素组；B组，重组Tet-on COLIA1基因腺病毒；C组，Tet-on空白腺病毒+强力霉素组；D组，空白对照组。

分别于注射腺病毒6、8周后在电子万能材料试验机上进行胫骨三点弯曲试验。

根据载荷-变形曲线计算骨折标本的生物力学参数，包括最大负荷、弹性模量、屈服点最大应力，同时采用micro-CT检测骨折愈合情况。

Porcine reproductive and respiratory syndrome virus as a vector:Immunogenicity of green fluorescent protein and porcine circovirus type 2capsid expressed from dedicated subgenomic RNAsYanlong Pei a ,Douglas C.Hodgins a ,Jiaqiang Wu a ,1,Siao-Kun W.Welch b ,Jay G.Calvert b ,Gang Li c ,Yijun Du d ,Cheng Song a ,d ,Dongwan Yoo d ,⁎aDepartment of Pathobiology,University of Guelph,Guelph,Ontario,Canada N1G 2W1bP fizer Animal Health,Kalamazoo,MI 49001,USA cInstitute of Animal Health and Husbandry,Chinese Academy of Agricultural Sciences,Beijing,China dDepartment of Pathobiology,University of Illinois at Urbana-Champaign,2001South Lincoln Ave,Urbana,IL 61802,USAa b s t r a c ta r t i c l e i n f o Article history:Received 31January 2009Returned to author for revision 3March 2009Accepted 31March 2009Available online 6May 2009Keywords:PRRSVReverse geneticsForeign gene expression vector Vaccine vector Nidovirus ArterivirusPorcine reproductive and respiratory syndrome virus (PRRSV)is the causative agent of PRRS,which is characterized by late-term abortions in sows and respiratory disease in young ing an infectious cDNA clone of North American PRRSV strain P129,the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes.Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6(TRS6)were inserted between ORFs 1b and 2a,yielding a general purpose expression vector.The enhanced green fluorescent protein (GFP)gene was cloned between the unique sites such that the inserted gene was transcribed from TRS2which was located upstream within ORF1b,while the copy of TRS6drives ORF2a/b transcription.Upon transfection of cells with this plasmid,PRRSV infection was initiated and progeny virus “P129-GFP ”was obtained.Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37serial in vitro passages.Subsequently,a capsid (C)protein gene was cloned from porcine circovirus type 2(PCV2)recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS)and inserted into the PRRSV infectious clone vector,generating virus “P129-PCV ”.To determine the immunogenicity of the recombinant viruses,pigs were immunized intramuscularly with P129-WT (wild-type),P129-GFP,or P129-PCV2.By 5weeks post-infection,speci fic antibody responses to GFP and PCV2capsid were elicited.This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens.©2009Elsevier Inc.All rights reserved.IntroductionPorcine reproductive and respiratory syndrome (PRRS)is an emerged and re-emerging disease in pigs.The disease was first recognized in Germany and the US almost simultaneously in the late 1980s and has since spread globally to most pork producing countries (Keffaber,1989;Ben field et al.,1992;Albina,1997).PRRS is characterized by abortions and mummi fied fetuses in sows,and respiratory distress with poor growth in young pigs.The disease is mild in gilts and boars,but the virus (PRRSV)persists in semen and thus can be transmitted widely by arti ficial insemination.Since its emergence,PRRS has become one of the most economically important diseases in the swine industry.Modi fied-live vaccines are available,but safety and limited ef ficacy areongoing concerns.No speci fic treatment is available for PRRS,and thus the economic losses are enormous.Numerous isolates of PRRSV representing many geographical regions have been sequenced,revea-ling the existence of two distinct genotypes of PRRSV:European (type I)and North American (type II).The two genotypes share overall sequence identity of 63%and differ antigenically as well as genetically (Nelson et al.,1993;Meng et al.,1995;Wootton et al.,2000).PRRS virus is an enveloped,single-stranded,positive-sense RNA virus belonging to the family Arteriviridae and along with the Coronaviridae family,forms the order Nidovirales .The PRRSV genome is approximately 15kb in size and includes the 5′cap structure and 3′polyadenylated tail (Sagripanti et al.,1986;Wootton et al.,2000).The genome consists of nine genes:open reading frames (ORFs)1a,1b,2a,2b,3,4,5,6,and 7.The 5′three-quarters of the genome consists of two slightly overlapping ORFs,1a and 1b,and they are translated directly from the genome-sense RNA.ORF1b is expressed as a fusion protein with ORF1a by a frame-shifting mechanism,and the ORF1a and ORFla/b proteins are auto-cleaved by viral proteases into at least 13cleavage products that are theVirology 389(2009)91–99⁎Corresponding author.E-mail address:dyoo@ (D.Yoo).1Current address:Shandong Key Laboratory of Animal Disease Control and Breeding,Shandong Academy of Agricultural Sciences,Jinan,Shandong,China.0042-6822/$–see front matter ©2009Elsevier Inc.All rights reserved.doi:10.1016/j.virol.2009.03.036Contents lists available at ScienceDirectVirologyj o u r n a l ho m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /y v i r onon-structural proteins Nsp1α,Nsp1β,and Nsp2through Nsp12.The Nsps are thought to be involved in genome replication and subgenome transcription(van Dinten et al.,1999;van Marle et al.,1999).The viral structural proteins are encoded by ORFs2a,2b,and3through7which are located downstream of ORFs1a and1b.These genes are expressed as a3′-coterminal nested set of subgenomic(sg)RNAs(de Vries et al., 1990).The5′untranslated leader sequences of the sgRNAs are derived from the5′end of the viral genome and fused to the body segments of the sgRNAs at conserved hexanucleotide motifs[5′UCAAC(U/C)3′] located immediate upstream of every transcription unit(de Vries et al., 1990;den Boon et al.,1996).The conserved hexanucleotide motif and poorly conservedflanking sequences form secondary structures in the sgRNAs that make up the transcriptional regulatory sequences(TRS)that are necessary for sgRNA formation(Pasternak et al.,2000).With the exception of sgRNA7,sgRNAs are structurally polycistronic but,with the exception of sgRNA2a/b,functionally monocistronic as only the5′most proximal gene of each sgRNA is translated.ORFs2a,2b,and3through7 code for GP2(glycoprotein2),E(envelope),GP3,GP4,GP5,M (membrane)and N(nucleocapsid)proteins,respectively,and they make up virion particles(Meulenberg et al.,1995).The recent development of infectious clones for PRRSV has allowed specific alterations of viral genomes and generation of mutant viruses(Yoo et al.,2004).However,manipulation of arterivirus genomes is complicated by the condensed organization of the viral genome.In the case of the European genotype of PRRSV,each gene overlaps slightly except for ORFs 1b and2a(Meulenberg et al.,1993),although the overlap of genes seems less important for virus replication and growth for equine arteritis virus (de Vries et al.,2000).For the North American genotype,structuralgenes Fig.1.Genomic organization of P129-WT,P129-GFP,and P129-PCV2.(A)Genomic organization and sequence of the region surrounding the ORF1b/ORF2junction of the unmodified “wild-type”P129strain of PRRSV(P129-WT).The boxed sequence indicates the core hexanucleotide of TRS2.Amino acids indicate translated sequences of polyprotein1a/b(ORF1b),the GP2protein(ORF2a),and the E protein(ORF2b).The initiation codons are indicated in bold,and the E protein sequence is italicized.Numbers in parenthesis indicate genomic sequence positions.Stars indicate translation stops.(B)P129-GFP.Unique AflII and Mlu I sites and a copy of TRS6were introduced into the non-coding region between ORF1b and ORF2a,creating expression vector pCMV-S-P129-1bMCS2.The GFP gene was amplified by PCR and inserted between the AflII and Mlu I restriction sites.(C)P129-PCV2.The PCV2 capsid gene was amplified by PCR and cloned between the AflII and Mlu I restriction sites.92Y.Pei et al./Virology389(2009)91–99also overlap with the exceptions of ORFs 1b and 2a,and ORFs 4and 5(Nelsen et al.,1999;den Boon et al.,1991;Snijder and Meulenberg,1998).Thus,the entire genome of the North American type PRRSV possesses only 4short non-coding regions:191nucleotides of 5′untranslated region (UTR),1nucleotide between ORF1b and ORF2a,10nucleotides between ORF4and ORF5,and 151nucleotides of 3′UTR upstream of the polyadenylation tail.The 5′and 3′UTRs contain genome replication and transcription signals,and therefore offer limited sites for gene insertion and manipulation.The presence of overlapping genes hampers mutational analysis of the N-and C-termini of the structural proteins and also makes it dif ficult to insert heterologous genes into the viral genome.In the present study,we used a genomic cDNA clone of PRRSV (Lee et al.,2005)to generate a vector for foreign gene expression from a dedicated subgenomic transcription unit inserted in the region between the structural and non-structural genes.The vector was used to express GFP in vitro and in vivo .The inserted gene was tolerated by the virus,stable for at least 37passages in cell culture,and induced antibodies to GFP in young pigs.Using this approach,we also generated a recombinant PRRSV expressing the capsid protein gene of porcine circovirus type 2(PCV2).PCV2is a small DNA virus in the Circoviridae family,with a genome ofonly 1.76kb.PCV2ORF1is essential for viral DNA replication (Mankertz et al.,1998;Fenaux et al.,2000),while ORF2encodes the capsid protein containing type-speci fic epitopes that are believed to be important for virus neutralization (Nawagitgul et al.,2000;Fenaux et al.,2004).Accumulating evidence suggests a major role for PCV2in postweaning multisystemic wasting syndrome (PMWS)and porcine dermatitis and nephropathy syndrome (PDNS)(Hasslung et al.,2005).These syndromes cause serious economic impacts in the swine industry today (Chae 2005).We showed the recombinant PRRSV P129-PCV2induced an anti-PCV2antibody response in immunized pigs in the presence of maternal antibodies to PCV2.Our vector construction may be applicable not only for PRRSV,but also for other members of the families Arteriviridae and Coronaviridae .ResultsDevelopment of PRRSV as an expression vector for GFPTo explore the possibility of developing PRRSV as a gene expression vector,the GFP gene was inserted between the stop codon ofORF1bFig.2.(A)Restriction patterns of P129-WT (lane 1),P129-GFP (lane 2),and P129-PCV2(lane 3)genomic clones generated by Sma I digestion.Fragments of 590bp,4736bp and 10,779bp are expected from all three clones.The fourth fragment varies in size depending on the gene inserted at the non-structural and structural gene junction.Relative to the P129-WT fragment (2787bp,lane 1),the GFP-containing fragment from P129-GFP is 766bp larger (3553bp,lane 2)and the PCV2capsid-containing fragment from P129-PCV2is 754bp larger (3541bp,lane 3).(B –E)Recovery of recombinant PRRSV foci from full-length genomic clones.(F)Integration of the GFP gene in P129-GFP viral genome.Genomic RNA was extracted from P129-GFP virus and digested with DNase I prior to reverse transcription.Without reverse transcription,no products were ampli fied from P129-GFP using ORF7-speci fic PCR primers P129-7F and P129-7R (lane 1)or primers P129-1bF and P129-2aR that span the insertion site (lane 2).With reverse transcription,primers P129-7F and P129-7R ampli fied the ORF 7fragment (534bp)from both P129-WT (lane 3)and P129-GFP (lane 4).Using P129-1bF and P129-2aR,a product of 766bp larger (lane 6)than the product from P129-WT (lane 5)was ampli fied from P129-GFP.(G)Integration of the PCV2C gene in PRRSV.Genomic RNA from P129-WT (lanes 9,10,12)and P129-PCV2(lanes 7,8,and 11)was ampli fied using primers P129-1bF and pared to P129-WT (lane 9)a product that is 754bp larger was ampli fied from P129-PCV2(lane 7).Using PCV2C gene speci fic primers PCV2-F and PCV2-R,the expected 497bp product was generated from P129-PCV2(lane 8)but not from P129-WT (lane 10).Using PRRSV ORF4-speci fic primers P129-4F and P129-4R,the expected 567bp product was ampli fied from both P129-PCV2and P129-WT (lanes 11and 12,respectively).93Y.Pei et al./Virology 389(2009)91–99and the start codon of ORF2a (Fig.1A).This non-coding region is extremely short,comprising only one adenosine nucleotide.The TRS associated with ORFs 2a and 2b (TRS2;TGAACC)is positioned 26nucleotides upstream from the start of ORF2a and is embedded in ORF1b.Upon insertion of GFP to the region,TRS2will drive transcription of the GFP gene instead of ORFs 2a and 2b.Thus,a synthetic TRS (TTAACC)with flanking sequences derived from TRS6was introduced 22nucleotides downstream of GFP and 17nucleotides upstream from the ORF2a start (Fig.1B).TRS6was chosen because RNA secondary structure suggested that it was shorter than other PRRSV TRS elements and because the distance between the copy of TRS6driving ORF2a/b and the authentic TRS6ensured that potential intramolecular homologous RNA recombination would result in a non-viable (ORF2-5deleted)virus.The PRRSV genomic clone containing GFP was designated P129-GFP,and the insertion was con firmed by restriction patterns (Fig.2A,lane 2)and sequencing.MARC-145cells were transfected with P129-GFP and the production of virus was monitored daily for development of cytopathic effect (CPE)(Figs.2B,C,D).CPE was observed 4days post-transfection and the development of CPE was one day slower than for P129-WT.After three consecutive passages,P129-GFP virus was re-examined for GFP sequence integration in the viral genome (Fig.2F).While ORF7ampli fication products were identical in size for P129-WT (lane 3)and P129-GFP (lane 4),1bF and 2aR primers produced a larger size product from P129-GFP (lane 6)than from P129-WT (lane 5).The larger product was the expected size for correct insertion of GFP gene intothe viral genome.Replication of P129-GFP was slightly slower than that of P129-WT at passages 1to 3.However,plaques were comparable in size and morphology for P129-WT and P129-GFP,and the titers at passage 3were 5×105plaque forming units (PFU)/ml and 1×105PFU/ml,respectively.Fluorescence was evident in P129-GFP-infected cells (Fig.3A),demonstrating the expression of GFP during infection.To examine the genetic stability of recombinant PRRSV,P129-GFP was passaged 37times in MARC-145cells and GFP expression was monitored by fluorescence microscopy.Individual plaques of 20formed by P129-GFP were randomly chosen and examined for fluorescence.The selected plaques were all positive for fluorescence (Table 1),and sequencing of the viral RNA con firmed stability of the insert (data not shown).This data showed the genetic stability of P129-GFP after serial passages in cell culture and the stable expression of GFP.It also demonstrates that the region between ORF1b and ORF2a is a suitable site for foreign gene insertion for PRRSV.This was the first demonstration of the use of a nidovirus as an expression vector wherein the foreign gene is inserted in the region between the non-structural and structural protein codingsequences.Fig.3.Expression of GFP or PCV2capsid protein by P129-GFP and P129-PCV2in MARC-145cells.(A)Live cells infected with P129-GFP passage 3;(B)live cells infected with P129-GFP passage 37;(C)uninfected cells;(D)P129-PCV2passage 3fixed and stained with PCV2-speci fic antibody conjugated with FITC at 48h post-infection;(E and F)P129-PCV2infected cells fixed and co-stained with PCV2-speci fic antibody conjugated with FITC (E)or GP4protein-speci fic monoclonal antibody 169(F).Table 1Stability of GFP expression in P129-GFP virus.Virus Titer (PFU/ml)GFP expressing plaques Passage 38.3×10e620positive/20plaques Passage 372.4×10e720positive/20plaques94Y.Pei et al./Virology 389(2009)91–99Construction of PRRSV expressing PCV2capsidPCV2is associated with porcine multisystemic wasting disease (PMWS,now termed PCVAD [PCV-associated disease])and porcine dermatitis and nephropathy syndrome (PDNS).PCV2is transmitted by the oro-nasal route and shed in the bronchial secretions and feces,thus the transmission route is similar to that of PRRSV.The capsid (C)protein is the major antigen able to elicit protective immunity against PCV2.Thus,using the PRRSV vector system described above,an additional recombinant PRRSV was constructed to carry the PCV2C gene.A 702bp C gene was cloned by PCR from a lung tissue positive for PCV2.Sequencing of the C gene showed 99–100%amino acid identity to published sequences available in the GenBank database.The P129-PCV2clone was constructed by inserting the C gene into PRRSV in the same way as the GFP gene insertion for P129-GFP (Fig.1C).The insertion of the C gene was con firmed by restriction digestion pattern (Fig.2A,lane 3)and sequencing.The P129-PCV2recombinant virus was recovered from MARC-145cells by transfection (Fig.2E),and the insertion of the C gene in the viral genome was con firmed by RT-PCR of the viral RNA (Fig.2G).The titer of passage 3virus was 2×105PFU/ml and the plaque morphology was indistinguishable from P129-WT.Infection of cells with P129-PCV2and staining with PCV2-speci fic antibody produced distinct fluorescence (Fig.3E),which shows the expression of the C protein by P129-PCV2.The capsid protein of PCV2is arginine-rich and normally shuttles into the nucleus during PCV2replication (Liu et al.,2001),and similarly,the PRRSV N has also been shown to localize in the nucleus and nucleolus (Lee et al.,2006;Pei et al.,2008).Thus,in cells infected with P129-PCV2,the synthesis of legitimate PCV2capsid should be evident by the translocation of capsid into the nucleus.Thus,the C protein expression by P129-PCV2was con firmed by co-staining of virus-infected cells with PRRSV N protein-speci fic antibody and PCV2-speci fic antiserum (Fig.4).While the PRRSV N protein was found in the both cytoplasm and the nucleolus as usual (panel A),the PCV2capsid protein was speci fically localized to the nucleus and nucleolus (panel C)in the same cell,clearly demonstrating the expression of PCV2capsid protein by the recombinant PRRSVP129-PCV2.Fig.4.Co-expression of the PRRSV N protein (green)and the PCV2capsid protein (red)during infection of P129-PCV2.MARC-145cells were infected with P129-PCV2and stained 24h post-infection with PRRSV N-speci fic MAb SDOW-17(A and B)or PCV2-speci fic pig serum (C and D).Arrows indicate the PRRSV N protein in the nucleolus (panel A)in addition to the cytoplasm and the PCV2capsid protein in the nucleus and nucleolus (panel C).Panel E shows the merge of A and C.Panel F shows the merge of B and D.Table 2PCR primers and their genomic Sequence (5′–3′)aGenomic position b PurposePCV2-F cacggatattgtagtcctggt 1093–1114PCV2PCR test PCV2-R ccgcaccttcggatatactgtc1565–1586PCV2PCR testPCV2-orf2F gatgcttaagatgacgtatccaaggtggcg 1715–1734PCV2ORF2ampli fication PCV2-orf2R gtacacgcgtcattaagggttaagtcccccc 1031–1050PCV2ORF2ampli ficationP129-F1F aacagaagagttgtcgggtccac11,699–11,721P12911,783–12,055,ampli fication P129-F1R gctttcacgcgtccccacttaagttcaattcaggcctaaagttggttca 12,031–12,055Introduction of A flII and Mlu IP129-F2F gcgacgcgt gttccgtggcaacccctttaaccagagtttcagcggaaga atgaaatggggtctatacaaagcctcttcgaca 12,056–12,089P12912,056–12,697,ampli fication and introduction of Mlu I and TRS6P129-F2R aacagaacggcacgatacaccacaaa 13,819–13,844P12912,056–12,679,ampli fication P129-7F tcatccgattgcggcaaatg 14,724–14,743P129ORF7ampli fication P129-7R agaatgccagcccatca 15,242–15,258P129ORF7ampli fication P129-4F gtttcacctagaatggctg 13,213–13,231ORF4ampli fication P129-4R ccccaacatacttgaacattc 13,750–13,770ORF4ampli ficationP129-1bF ggtgaggactgggaggattac 11,921–11,941ORF1b –ORF2a,region ampli fication P129-2aRcagtacgtagcattggaacc12,758–12,777ORF1b –ORF2a,region ampli ficationa Restriction sites are underlined.TRS6and surrounding sequences are indicated in bold.bGenomic positions for PCV2primers were based on GenBank accession AF027217.Genomic position for P129primers were based on GenBank accession AF494042.95Y.Pei et al./Virology 389(2009)91–99Infection of pigs and antibody responses to GFP and PCV2capsid protein To determine antibody responses to GFP and the PCV2capsid protein,pigs were immunized with the recombinant PRRS viruses.Fifteen PRRSV-free pigs at 4weeks of age were randomly allotted to 3groups of 5pigs each.Animals were immunized twice on days 0and 21by intramuscular injection of 5×105PFU per animal with either P129-WT,P129-GFP,or P129-PCV2.Following inoculation,the animalswere maintained for 5weeks for clinical observation and serum collection.Clinical signs of PRRS were minimal and comparable in all 3groups (data not shown).Mild clinical signs were not unexpected,since the infectious cDNA clone used in these studies was not attenuated.Tonsil samples were collected at necropsy on day 35and assessed by RT-PCR for the presence of PRRSV ORF7using primers P129-7F and P129-7R (Table 2)as well as the GFP and PCV2capsid inserts using primers P129-1bF and P129-2aR.All 15pigs were positive for ORF7,indicating infection and persistence of PRRSV in the tonsils.PCR products from the ORF1b/ORF2a junction were not detected in these tonsil samples,possibly due to the much lower molar ratio of ORF1b-containing RNA template relative to ORF7-containing RNA in infected cells.Alternatively,it is possible that the GFP and PCV2capsid genes might have been unstable in vivo and lost in the inoculated pigs over time.To examine antibody responses in these pigs,ELISAs were conducted for PRRSV,GFP,and PCV2C protein.All pigs produced good levels of antibodies to PRRSV (Fig.5A),and the antibody titers were comparable among groups.P129-GFP elicited speci fic antibodies to GFP,whereas pigs immunized with P129-PCV2or P129-WT were negative for GFP (Fig.5B).The GFP antibodies increased following first immunization,and the second immunization at day 21boosted the antibody response somewhat (Fig.5B).Similarly,antibodies for PCV2C protein were detected in pigs immunized with P129-PCV2(Fig.5C).In these pigs,however,PCV2antibodies were detected at day 0in all 3treatment groups and tended to wane over time.These antibodies likely represent maternal antibodies taken up in colostrum shortly after farrowing at the farm of origin and prior to experimental infection.However,an increase in anti-PCV2antibodies was observed at 28days in the P129-PCV2group only (Fig.5C)due to boosting effects from the second immunization at day 21.In contrast,anti-PCV2antibodies in the other two treatment groups waned gradually from day 0until at least until day 35.To further determine the speci fic antibody responses to GFP and the PCV2C protein,Western blots were conducted using sera from these pigs.Serum from the P129-GFP group was reactive with GFP (Fig.6A),consistent with the ELISA data (Fig.5B).Similarly,serum from the P129-PCV2group showed a strong reaction with C pro-tein (Fig.6B).Weaker reactions were observed using sera from the P129-WT and P129-GFP groups,consistent with the presence of maternalantibodies.Fig.5.ELISA showing induction of speci fic antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT viruses.(A)antibodies to PRRSV N protein;(B)antibodies to GFP;(C)antibodies to PCV2Cprotein.Fig. 6.Western blots showing induction of speci fic antibodies in sera from pigs inoculated with P129-GFP,P129-PCV2,or P129-WT at day 0or day 35post-inoculation.Blots contain GFP protein (A)or PCV2virions (B).Arrows indicate positions of GFP and PCV2capsid protein.C denotes positive control (anti-GFP monoclonal antibody).M indicates molecular weight markers.The virus used to infect pigs that contributed to the serum pools is indicated above each lane.96Y.Pei et al./Virology 389(2009)91–99DiscussionThe primary target cell of PRRSV is the alveolar macrophage,and pigs are the only animal species known to be susceptible to PRRSV infection.Therefore,development of PRRSV as a vaccine vector would be useful for the delivery of porcine pathogen genes to the respiratory tract of the pig.Arterivirus genomes are organized in a complex way. Most genes overlap one another in different reading frames,making it difficult to engineer the genome for foreign gene insertion.An early approach involved engineering the3′terminal region of the N gene (Groot Bramel-Verheije et al.,2000).Modification of the3′terminal sequence of N gene was possible and caused only minimal effects on virus replication and growth.However,no more than7amino acids were inserted.More recently,Nsp2,a large product of proteolytic cleavage of the ORF1a and ORF1a/b polyproteins,was found to be remarkably heterogeneous in sequence,with several hypervariable regions.Therefore,the GFP gene was inserted in-frame into or between the hypervariable regions to create nsp2-GFP fusion proteins (Fang et al.,2006;Kim et al.,2007).This approach was successful and allowed for the production of recombinant viruses.However,in all cases the inserted GFP gene was not phenotypically stable and lost greenfluorescence after several passages in cell culture(Fang et al., 2006;Kim et al.,2007;Han et al.,2007).In the present study,we inserted GFP and the PCV2capsid genes into the short region separating the non-structural protein genes from the structural protein genes.In contrast to the nsp2-GFP fusion proteins described above,we expressed GFP as a separate transcription unit resulting in an additional sgRNA.This approach has the advantage of eliminating the need to alter the coding sequence of a viral gene,and also minimizes effects on expression and post-translational modification of viral gene products.As a result,our recombinant virus was stable for at least37cell culture passages without loss of the gene or the greenfluorescent phenotype.PRRSV is known to induce immune suppressive effects in pigs (Charerntantanakul et al.,2006)and can persist up to6months in infected pigs(Wills et al.,1997).For these reasons,co-infection of PRRSV with other pathogen such as PCV2can result in much more severe clinical outcome than either agent alone(Harms et al.,2001; Kim et al.,2003).Therefore,a dual-purpose vaccine capable of protecting pigs against both PRRS and PCV2would be advantageous. Our study demonstrates the potential of PRRSV as a viral vaccine vector.Prior to the purchase of animals for infection studies using P129-PCV2,all pigs were screened for PCV2by PCR.PCR is the gold standard for detection of PCV2,whereas antibody screening is not routinely conducted in diagnostic laboratories due to cross-reactivity between PCV2and PCV1which is ubiquitous and widely distributed in thefield (Magar et al.,2000).Although all pigs entering the present study tested negative for the presence of PCV2by PCR,antibodies were detected in sera collected on day0.These antibodies were most likely of maternal origin,since they decreased in concentration over the duration of the experiment in pigs receiving P129-WT or P129-GFP. Serological studies show that maternal antibodies for PCV2decay during thefirst2months of life(Rodríguez et al.,2002;Larochelle et al.,2003).Western blots and ELISA gave comparable results in this regard.In the current study,antibodies to PCV2only increased after day21of the study and did so only in pigs receiving P129-PCV2, suggesting that the increases were most likely specific responses to P129-PCV2vaccination.At the termination of the study(day35post-infection),tonsil samples in all3groups were positive for the PRRSV N gene by RT-PCR.The presence of the inserted GFP and PCV2genes in these same samples could not be confirmed using primersflanking the region of the gene insertion.No products were amplified,even from pigs infected with the P129-WT virus.Failure to amplify the ORF1b/ORF2a junction is likely the result of template RNA concentra-tions below the level of detection of the PCR assay,and is consistent with the observation that the copy number of ORF1b(present only on genomic RNA)is much lower than the copy number of ORF7(present on all sgRNAs as well as genomic RNA).In conclusion,a PRRSV gene expression vector was generated, capable of expressing a foreign gene from an additional transcription unit located in the region between the non-structural and structural genes of the virus.The recombinant PRRSVs expressing the GFP or PCV2capsid genes were generated and shown to replicate well in cell culture.The addition of766nt(GFP)or754nt(PCV2capsid)of foreign genetic material,representing approximately5%of the PRRSV genome,was tolerated with no evidence of compensatory deletions or rearrangements elsewhere in the genome.Pigs inoculated with these recombinant PRRSVs produced foreign gene specific antibodies.Our study demonstrates the potential of PRRSV to function as a vector for development of multivalent vaccines against swine diseases.Materials and methodsCells and virusesMARC-145African green monkey kidney cells(Kim et al.,1993) were maintained as previously described(Lee et al.,2003).Dulac porcine kidney cells,kindly provided by L.Babiuk(Vaccine and Infectious Disease Organization,SK,Canada),were grown in Modified Eagle's Medium(MEM)supplemented with5%fetal bovine serum (FBS;Gibco BRL),penicillin(100U/ml),and streptomycin(50μg/ml). Cells were maintained at37°C with5%CO2.Stocks of recombinant viruses derived from infectious clones were prepared by passaging three times on MARC-145cells.Titers of PRRSV were determined by standard plaque assays on MARC-145cells using6-well plates(35mm diameter)in duplicate.Plaques were stained with0.01%neutral red. For isolation of PCV2,lung tissues were obtained from a PCR-positive pig(Ontario18099)submitted to Animal Health Laboratory of the University of Guelph(Guelph,ON,Canada).The tissues were homogenized in PBS and thefiltrate was used to infect Dulac cells. At3days post-infection,cells were stained using a porcine circovirus hyperimmune serum(VMRD,Pullman,WA,USA)to confirm infection. On day4post-infection,cells were harvested and freeze–thawed three times.Cell debris was removed by centrifugation at5000×g, and the supernatant was stored at−80°C until use.Construction of a PRRSV expression vector and recombinant PRRSVsFor construction of the PRRSV expression vector,the regions flanking the ORF1b/ORF2a junction were amplified by PCR using the shuttle plasmid p2-7D4(containing genomic positions11,504to 15,395)as template.Two DNA products corresponding to positions 11,783to12,055and12,056to12,697were amplified.The primer set P129F1-F(containing an Eco47III site)and P129F1-R(containing AflII and Mlu I sites)was used for the upstream product(Table2).The primers set P129F2-F(containing Mlu I site and TRS6)and P129F2-R (containing a Bsr GI site)was used to amplify the downstream product(Table2).The twoflanking products were digested with Eco 47III–Mlu I and Mlu I–Bsr GI,respectively,and included in a three-way ligation with Eco47III–Bsr GI-digested full-length genomic cDNA clone pCMV-S-P129(Lee et al.,2003).The resulting construct pCMV-S-P129-1bMCS2contained a complete PRRSV genome with unique AflII and Mlu I sites and a copy of TRS6inserted between ORF1b and ORF2a.Transfection of MARC-145cells with this construct produced viable virus that replicated normally(data not shown).For insertion of foreign genes,pCMV-S-P129-1bMCS2was digested with AflII–Mlu I and ligated to either the GFP gene or the PCV2capsid gene into which AflII and Mlu I sites were introduced during PCR (Fig.1).Recombinant genomic clones were screened by Sma I digestion(Fig.2),and selected clones were sequenced to confirm the presence of insertions.The PCV2capsid protein gene was cloned97Y.Pei et al./Virology389(2009)91–99。

DOI: 10.1126/science.1094786, 441 (2004);304Science et al.Mitchell S. Abrahamsen,Cryptosporidium parvum Complete Genome Sequence of the Apicomplexan, (this information is current as of October 7, 2009 ):The following resources related to this article are available online at/cgi/content/full/304/5669/441version of this article at:including high-resolution figures, can be found in the online Updated information and services,/cgi/content/full/1094786/DC1 can be found at:Supporting Online Material/cgi/content/full/304/5669/441#otherarticles , 9 of which can be accessed for free: cites 25 articles This article 239 article(s) on the ISI Web of Science. cited by This article has been /cgi/content/full/304/5669/441#otherarticles 53 articles hosted by HighWire Press; see: cited by This article has been/cgi/collection/genetics Genetics: subject collections This article appears in the following/about/permissions.dtl in whole or in part can be found at: this article permission to reproduce of this article or about obtaining reprints Information about obtaining registered trademark of AAAS.is a Science 2004 by the American Association for the Advancement of Science; all rights reserved. The title Copyright American Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005. (print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by the Science o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m3.R.Jackendoff,Foundations of Language:Brain,Gram-mar,Evolution(Oxford Univ.Press,Oxford,2003).4.Although for Frege(1),reference was established rela-tive to objects in the world,here we follow Jackendoff’s suggestion(3)that this is done relative to objects and the state of affairs as mentally represented.5.S.Zola-Morgan,L.R.Squire,in The Development andNeural Bases of Higher Cognitive Functions(New York Academy of Sciences,New York,1990),pp.434–456.6.N.Chomsky,Reflections on Language(Pantheon,New York,1975).7.J.Katz,Semantic Theory(Harper&Row,New York,1972).8.D.Sperber,D.Wilson,Relevance(Harvard Univ.Press,Cambridge,MA,1986).9.K.I.Forster,in Sentence Processing,W.E.Cooper,C.T.Walker,Eds.(Erlbaum,Hillsdale,NJ,1989),pp.27–85.10.H.H.Clark,Using Language(Cambridge Univ.Press,Cambridge,1996).11.Often word meanings can only be fully determined byinvokingworld knowledg e.For instance,the meaningof “flat”in a“flat road”implies the absence of holes.However,in the expression“aflat tire,”it indicates the presence of a hole.The meaningof“finish”in the phrase “Billfinished the book”implies that Bill completed readingthe book.However,the phrase“the g oatfin-ished the book”can only be interpreted as the goat eatingor destroyingthe book.The examples illustrate that word meaningis often underdetermined and nec-essarily intertwined with general world knowledge.In such cases,it is hard to see how the integration of lexical meaning and general world knowledge could be strictly separated(3,31).12.W.Marslen-Wilson,C.M.Brown,L.K.Tyler,Lang.Cognit.Process.3,1(1988).13.ERPs for30subjects were averaged time-locked to theonset of the critical words,with40items per condition.Sentences were presented word by word on the centerof a computer screen,with a stimulus onset asynchronyof600ms.While subjects were readingthe sentences,their EEG was recorded and amplified with a high-cut-off frequency of70Hz,a time constant of8s,and asamplingfrequency of200Hz.14.Materials and methods are available as supportingmaterial on Science Online.15.M.Kutas,S.A.Hillyard,Science207,203(1980).16.C.Brown,P.Hagoort,J.Cognit.Neurosci.5,34(1993).17.C.M.Brown,P.Hagoort,in Architectures and Mech-anisms for Language Processing,M.W.Crocker,M.Pickering,C.Clifton Jr.,Eds.(Cambridge Univ.Press,Cambridge,1999),pp.213–237.18.F.Varela et al.,Nature Rev.Neurosci.2,229(2001).19.We obtained TFRs of the single-trial EEG data by con-volvingcomplex Morlet wavelets with the EEG data andcomputingthe squared norm for the result of theconvolution.We used wavelets with a7-cycle width,with frequencies ranging from1to70Hz,in1-Hz steps.Power values thus obtained were expressed as a per-centage change relative to the power in a baselineinterval,which was taken from150to0ms before theonset of the critical word.This was done in order tonormalize for individual differences in EEG power anddifferences in baseline power between different fre-quency bands.Two relevant time-frequency compo-nents were identified:(i)a theta component,rangingfrom4to7Hz and from300to800ms after wordonset,and(ii)a gamma component,ranging from35to45Hz and from400to600ms after word onset.20.C.Tallon-Baudry,O.Bertrand,Trends Cognit.Sci.3,151(1999).tner et al.,Nature397,434(1999).22.M.Bastiaansen,P.Hagoort,Cortex39(2003).23.O.Jensen,C.D.Tesche,Eur.J.Neurosci.15,1395(2002).24.Whole brain T2*-weighted echo planar imaging bloodoxygen level–dependent(EPI-BOLD)fMRI data wereacquired with a Siemens Sonata1.5-T magnetic reso-nance scanner with interleaved slice ordering,a volumerepetition time of2.48s,an echo time of40ms,a90°flip angle,31horizontal slices,a64ϫ64slice matrix,and isotropic voxel size of3.5ϫ3.5ϫ3.5mm.For thestructural magnetic resonance image,we used a high-resolution(isotropic voxels of1mm3)T1-weightedmagnetization-prepared rapid gradient-echo pulse se-quence.The fMRI data were preprocessed and analyzedby statistical parametric mappingwith SPM99software(http://www.fi/spm99).25.S.E.Petersen et al.,Nature331,585(1988).26.B.T.Gold,R.L.Buckner,Neuron35,803(2002).27.E.Halgren et al.,J.Psychophysiol.88,1(1994).28.E.Halgren et al.,Neuroimage17,1101(2002).29.M.K.Tanenhaus et al.,Science268,1632(1995).30.J.J.A.van Berkum et al.,J.Cognit.Neurosci.11,657(1999).31.P.A.M.Seuren,Discourse Semantics(Basil Blackwell,Oxford,1985).32.We thank P.Indefrey,P.Fries,P.A.M.Seuren,and M.van Turennout for helpful discussions.Supported bythe Netherlands Organization for Scientific Research,grant no.400-56-384(P.H.).Supporting Online Material/cgi/content/full/1095455/DC1Materials and MethodsFig.S1References and Notes8January2004;accepted9March2004Published online18March2004;10.1126/science.1095455Include this information when citingthis paper.Complete Genome Sequence ofthe Apicomplexan,Cryptosporidium parvumMitchell S.Abrahamsen,1,2*†Thomas J.Templeton,3†Shinichiro Enomoto,1Juan E.Abrahante,1Guan Zhu,4 Cheryl ncto,1Mingqi Deng,1Chang Liu,1‡Giovanni Widmer,5Saul Tzipori,5GregoryA.Buck,6Ping Xu,6 Alan T.Bankier,7Paul H.Dear,7Bernard A.Konfortov,7 Helen F.Spriggs,7Lakshminarayan Iyer,8Vivek Anantharaman,8L.Aravind,8Vivek Kapur2,9The apicomplexan Cryptosporidium parvum is an intestinal parasite that affects healthy humans and animals,and causes an unrelenting infection in immuno-compromised individuals such as AIDS patients.We report the complete ge-nome sequence of C.parvum,type II isolate.Genome analysis identifies ex-tremely streamlined metabolic pathways and a reliance on the host for nu-trients.In contrast to Plasmodium and Toxoplasma,the parasite lacks an api-coplast and its genome,and possesses a degenerate mitochondrion that has lost its genome.Several novel classes of cell-surface and secreted proteins with a potential role in host interactions and pathogenesis were also detected.Elu-cidation of the core metabolism,including enzymes with high similarities to bacterial and plant counterparts,opens new avenues for drug development.Cryptosporidium parvum is a globally impor-tant intracellular pathogen of humans and animals.The duration of infection and patho-genesis of cryptosporidiosis depends on host immune status,ranging from a severe but self-limiting diarrhea in immunocompetent individuals to a life-threatening,prolonged infection in immunocompromised patients.Asubstantial degree of morbidity and mortalityis associated with infections in AIDS pa-tients.Despite intensive efforts over the past20years,there is currently no effective ther-apy for treating or preventing C.parvuminfection in humans.Cryptosporidium belongs to the phylumApicomplexa,whose members share a com-mon apical secretory apparatus mediating lo-comotion and tissue or cellular invasion.Many apicomplexans are of medical or vet-erinary importance,including Plasmodium,Babesia,Toxoplasma,Neosprora,Sarcocys-tis,Cyclospora,and Eimeria.The life cycle ofC.parvum is similar to that of other cyst-forming apicomplexans(e.g.,Eimeria and Tox-oplasma),resulting in the formation of oocysts1Department of Veterinary and Biomedical Science,College of Veterinary Medicine,2Biomedical Genom-ics Center,University of Minnesota,St.Paul,MN55108,USA.3Department of Microbiology and Immu-nology,Weill Medical College and Program in Immu-nology,Weill Graduate School of Medical Sciences ofCornell University,New York,NY10021,USA.4De-partment of Veterinary Pathobiology,College of Vet-erinary Medicine,Texas A&M University,College Sta-tion,TX77843,USA.5Division of Infectious Diseases,Tufts University School of Veterinary Medicine,NorthGrafton,MA01536,USA.6Center for the Study ofBiological Complexity and Department of Microbiol-ogy and Immunology,Virginia Commonwealth Uni-versity,Richmond,VA23198,USA.7MRC Laboratoryof Molecular Biology,Hills Road,Cambridge CB22QH,UK.8National Center for Biotechnology Infor-mation,National Library of Medicine,National Insti-tutes of Health,Bethesda,MD20894,USA.9Depart-ment of Microbiology,University of Minnesota,Min-neapolis,MN55455,USA.*To whom correspondence should be addressed.E-mail:abe@†These authors contributed equally to this work.‡Present address:Bioinformatics Division,Genetic Re-search,GlaxoSmithKline Pharmaceuticals,5MooreDrive,Research Triangle Park,NC27009,USA.R E P O R T S SCIENCE VOL30416APRIL2004441o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mthat are shed in the feces of infected hosts.C.parvum oocysts are highly resistant to environ-mental stresses,including chlorine treatment of community water supplies;hence,the parasite is an important water-and food-borne pathogen (1).The obligate intracellular nature of the par-asite ’s life cycle and the inability to culture the parasite continuously in vitro greatly impair researchers ’ability to obtain purified samples of the different developmental stages.The par-asite cannot be genetically manipulated,and transformation methodologies are currently un-available.To begin to address these limitations,we have obtained the complete C.parvum ge-nome sequence and its predicted protein com-plement.(This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the project accession AAEE00000000.The version described in this paper is the first version,AAEE01000000.)The random shotgun approach was used to obtain the complete DNA sequence (2)of the Iowa “type II ”isolate of C.parvum .This isolate readily transmits disease among numerous mammals,including humans.The resulting ge-nome sequence has roughly 13ϫgenome cov-erage containing five gaps and 9.1Mb of totalDNA sequence within eight chromosomes.The C.parvum genome is thus quite compact rela-tive to the 23-Mb,14-chromosome genome of Plasmodium falciparum (3);this size difference is predominantly the result of shorter intergenic regions,fewer introns,and a smaller number of genes (Table 1).Comparison of the assembled sequence of chromosome VI to that of the recently published sequence of chromosome VI (4)revealed that our assembly contains an ad-ditional 160kb of sequence and a single gap versus two,with the common sequences dis-playing a 99.993%sequence identity (2).The relative paucity of introns greatly simplified gene predictions and facilitated an-notation (2)of predicted open reading frames (ORFs).These analyses provided an estimate of 3807protein-encoding genes for the C.parvum genome,far fewer than the estimated 5300genes predicted for the Plasmodium genome (3).This difference is primarily due to the absence of an apicoplast and mitochondrial genome,as well as the pres-ence of fewer genes encoding metabolic functions and variant surface proteins,such as the P.falciparum var and rifin molecules (Table 2).An analysis of the encoded pro-tein sequences with the program SEG (5)shows that these protein-encoding genes are not enriched in low-complexity se-quences (34%)to the extent observed in the proteins from Plasmodium (70%).Our sequence analysis indicates that Cryptosporidium ,unlike Plasmodium and Toxoplasma ,lacks both mitochondrion and apicoplast genomes.The overall complete-ness of the genome sequence,together with the fact that similar DNA extraction proce-dures used to isolate total genomic DNA from C.parvum efficiently yielded mito-chondrion and apicoplast genomes from Ei-meria sp.and Toxoplasma (6,7),indicates that the absence of organellar genomes was unlikely to have been the result of method-ological error.These conclusions are con-sistent with the absence of nuclear genes for the DNA replication and translation machinery characteristic of mitochondria and apicoplasts,and with the lack of mito-chondrial or apicoplast targeting signals for tRNA synthetases.A number of putative mitochondrial pro-teins were identified,including components of a mitochondrial protein import apparatus,chaperones,uncoupling proteins,and solute translocators (table S1).However,the ge-nome does not encode any Krebs cycle en-zymes,nor the components constituting the mitochondrial complexes I to IV;this finding indicates that the parasite does not rely on complete oxidation and respiratory chains for synthesizing adenosine triphosphate (ATP).Similar to Plasmodium ,no orthologs for the ␥,␦,or εsubunits or the c subunit of the F 0proton channel were detected (whereas all subunits were found for a V-type ATPase).Cryptosporidium ,like Eimeria (8)and Plas-modium ,possesses a pyridine nucleotide tran-shydrogenase integral membrane protein that may couple reduced nicotinamide adenine dinucleotide (NADH)and reduced nico-tinamide adenine dinucleotide phosphate (NADPH)redox to proton translocation across the inner mitochondrial membrane.Unlike Plasmodium ,the parasite has two copies of the pyridine nucleotide transhydrogenase gene.Also present is a likely mitochondrial membrane –associated,cyanide-resistant alter-native oxidase (AOX )that catalyzes the reduction of molecular oxygen by ubiquinol to produce H 2O,but not superoxide or H 2O 2.Several genes were identified as involved in biogenesis of iron-sulfur [Fe-S]complexes with potential mitochondrial targeting signals (e.g.,nifS,nifU,frataxin,and ferredoxin),supporting the presence of a limited electron flux in the mitochondrial remnant (table S2).Our sequence analysis confirms the absence of a plastid genome (7)and,additionally,the loss of plastid-associated metabolic pathways including the type II fatty acid synthases (FASs)and isoprenoid synthetic enzymes thatTable 1.General features of the C.parvum genome and comparison with other single-celled eukaryotes.Values are derived from respective genome project summaries (3,26–28).ND,not determined.FeatureC.parvum P.falciparum S.pombe S.cerevisiae E.cuniculiSize (Mbp)9.122.912.512.5 2.5(G ϩC)content (%)3019.43638.347No.of genes 38075268492957701997Mean gene length (bp)excluding introns 1795228314261424ND Gene density (bp per gene)23824338252820881256Percent coding75.352.657.570.590Genes with introns (%)553.9435ND Intergenic regions (G ϩC)content %23.913.632.435.145Mean length (bp)5661694952515129RNAsNo.of tRNA genes 454317429944No.of 5S rRNA genes 6330100–2003No.of 5.8S ,18S ,and 28S rRNA units 57200–400100–20022Table parison between predicted C.parvum and P.falciparum proteins.FeatureC.parvum P.falciparum *Common †Total predicted proteins380752681883Mitochondrial targeted/encoded 17(0.45%)246(4.7%)15Apicoplast targeted/encoded 0581(11.0%)0var/rif/stevor ‡0236(4.5%)0Annotated as protease §50(1.3%)31(0.59%)27Annotated as transporter ࿣69(1.8%)34(0.65%)34Assigned EC function ¶167(4.4%)389(7.4%)113Hypothetical proteins925(24.3%)3208(60.9%)126*Values indicated for P.falciparum are as reported (3)with the exception of those for proteins annotated as protease or transporter.†TBLASTN hits (e Ͻ–5)between C.parvum and P.falciparum .‡As reported in (3).§Pre-dicted proteins annotated as “protease or peptidase”for C.parvum (CryptoGenome database,)and P.falciparum (PlasmoDB database,).࿣Predicted proteins annotated as “trans-porter,permease of P-type ATPase”for C.parvum (CryptoGenome)and P.falciparum (PlasmoDB).¶Bidirectional BLAST hit (e Ͻ–15)to orthologs with assigned Enzyme Commission (EC)numbers.Does not include EC assignment numbers for protein kinases or protein phosphatases (due to inconsistent annotation across genomes),or DNA polymerases or RNA polymerases,as a result of issues related to subunit inclusion.(For consistency,46proteins were excluded from the reported P.falciparum values.)R E P O R T S16APRIL 2004VOL 304SCIENCE 442 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o mare otherwise localized to the plastid in other apicomplexans.C.parvum fatty acid biosynthe-sis appears to be cytoplasmic,conducted by a large(8252amino acids)modular type I FAS (9)and possibly by another large enzyme that is related to the multidomain bacterial polyketide synthase(10).Comprehensive screening of the C.parvum genome sequence also did not detect orthologs of Plasmodium nuclear-encoded genes that contain apicoplast-targeting and transit sequences(11).C.parvum metabolism is greatly stream-lined relative to that of Plasmodium,and in certain ways it is reminiscent of that of another obligate eukaryotic parasite,the microsporidian Encephalitozoon.The degeneration of the mi-tochondrion and associated metabolic capabili-ties suggests that the parasite largely relies on glycolysis for energy production.The parasite is capable of uptake and catabolism of mono-sugars(e.g.,glucose and fructose)as well as synthesis,storage,and catabolism of polysac-charides such as trehalose and amylopectin. Like many anaerobic organisms,it economizes ATP through the use of pyrophosphate-dependent phosphofructokinases.The conver-sion of pyruvate to acetyl–coenzyme A(CoA) is catalyzed by an atypical pyruvate-NADPH oxidoreductase(Cp PNO)that contains an N-terminal pyruvate–ferredoxin oxidoreductase (PFO)domain fused with a C-terminal NADPH–cytochrome P450reductase domain (CPR).Such a PFO-CPR fusion has previously been observed only in the euglenozoan protist Euglena gracilis(12).Acetyl-CoA can be con-verted to malonyl-CoA,an important precursor for fatty acid and polyketide biosynthesis.Gly-colysis leads to several possible organic end products,including lactate,acetate,and ethanol. The production of acetate from acetyl-CoA may be economically beneficial to the parasite via coupling with ATP production.Ethanol is potentially produced via two in-dependent pathways:(i)from the combination of pyruvate decarboxylase and alcohol dehy-drogenase,or(ii)from acetyl-CoA by means of a bifunctional dehydrogenase(adhE)with ac-etaldehyde and alcohol dehydrogenase activi-ties;adhE first converts acetyl-CoA to acetal-dehyde and then reduces the latter to ethanol. AdhE predominantly occurs in bacteria but has recently been identified in several protozoans, including vertebrate gut parasites such as Enta-moeba and Giardia(13,14).Adjacent to the adhE gene resides a second gene encoding only the AdhE C-terminal Fe-dependent alcohol de-hydrogenase domain.This gene product may form a multisubunit complex with AdhE,or it may function as an alternative alcohol dehydro-genase that is specific to certain growth condi-tions.C.parvum has a glycerol3-phosphate dehydrogenase similar to those of plants,fungi, and the kinetoplastid Trypanosoma,but(unlike trypanosomes)the parasite lacks an ortholog of glycerol kinase and thus this pathway does not yield glycerol production.In addition to themodular fatty acid synthase(Cp FAS1)andpolyketide synthase homolog(Cp PKS1), C.parvum possesses several fatty acyl–CoA syn-thases and a fatty acyl elongase that may partici-pate in fatty acid metabolism.Further,enzymesfor the metabolism of complex lipids(e.g.,glyc-erolipid and inositol phosphate)were identified inthe genome.Fatty acids are apparently not anenergy source,because enzymes of the fatty acidoxidative pathway are absent,with the exceptionof a3-hydroxyacyl-CoA dehydrogenase.C.parvum purine metabolism is greatlysimplified,retaining only an adenosine ki-nase and enzymes catalyzing conversionsof adenosine5Ј-monophosphate(AMP)toinosine,xanthosine,and guanosine5Ј-monophosphates(IMP,XMP,and GMP).Among these enzymes,IMP dehydrogenase(IMPDH)is phylogenetically related toε-proteobacterial IMPDH and is strikinglydifferent from its counterparts in both thehost and other apicomplexans(15).In con-trast to other apicomplexans such as Toxo-plasma gondii and P.falciparum,no geneencoding hypoxanthine-xanthineguaninephosphoribosyltransferase(HXGPRT)is de-tected,in contrast to a previous report on theactivity of this enzyme in C.parvum sporo-zoites(16).The absence of HXGPRT sug-gests that the parasite may rely solely on asingle enzyme system including IMPDH toproduce GMP from AMP.In contrast to otherapicomplexans,the parasite appears to relyon adenosine for purine salvage,a modelsupported by the identification of an adeno-sine transporter.Unlike other apicomplexansand many parasitic protists that can synthe-size pyrimidines de novo,C.parvum relies onpyrimidine salvage and retains the ability forinterconversions among uridine and cytidine5Ј-monophosphates(UMP and CMP),theirdeoxy forms(dUMP and dCMP),and dAMP,as well as their corresponding di-and triphos-phonucleotides.The parasite has also largelyshed the ability to synthesize amino acids denovo,although it retains the ability to convertselect amino acids,and instead appears torely on amino acid uptake from the host bymeans of a set of at least11amino acidtransporters(table S2).Most of the Cryptosporidium core pro-cesses involved in DNA replication,repair,transcription,and translation conform to thebasic eukaryotic blueprint(2).The transcrip-tional apparatus resembles Plasmodium interms of basal transcription machinery.How-ever,a striking numerical difference is seenin the complements of two RNA bindingdomains,Sm and RRM,between P.falcipa-rum(17and71domains,respectively)and C.parvum(9and51domains).This reductionresults in part from the loss of conservedproteins belonging to the spliceosomal ma-chinery,including all genes encoding Smdomain proteins belonging to the U6spliceo-somal particle,which suggests that this par-ticle activity is degenerate or entirely lost.This reduction in spliceosomal machinery isconsistent with the reduced number of pre-dicted introns in Cryptosporidium(5%)rela-tive to Plasmodium(Ͼ50%).In addition,keycomponents of the small RNA–mediatedposttranscriptional gene silencing system aremissing,such as the RNA-dependent RNApolymerase,Argonaute,and Dicer orthologs;hence,RNA interference–related technolo-gies are unlikely to be of much value intargeted disruption of genes in C.parvum.Cryptosporidium invasion of columnarbrush border epithelial cells has been de-scribed as“intracellular,but extracytoplas-mic,”as the parasite resides on the surface ofthe intestinal epithelium but lies underneaththe host cell membrane.This niche may al-low the parasite to evade immune surveil-lance but take advantage of solute transportacross the host microvillus membrane or theextensively convoluted parasitophorous vac-uole.Indeed,Cryptosporidium has numerousgenes(table S2)encoding families of putativesugar transporters(up to9genes)and aminoacid transporters(11genes).This is in starkcontrast to Plasmodium,which has fewersugar transporters and only one putative ami-no acid transporter(GenBank identificationnumber23612372).As a first step toward identification ofmulti–drug-resistant pumps,the genome se-quence was analyzed for all occurrences ofgenes encoding multitransmembrane proteins.Notable are a set of four paralogous proteinsthat belong to the sbmA family(table S2)thatare involved in the transport of peptide antibi-otics in bacteria.A putative ortholog of thePlasmodium chloroquine resistance–linkedgene Pf CRT(17)was also identified,althoughthe parasite does not possess a food vacuole likethe one seen in Plasmodium.Unlike Plasmodium,C.parvum does notpossess extensive subtelomeric clusters of anti-genically variant proteins(exemplified by thelarge families of var and rif/stevor genes)thatare involved in immune evasion.In contrast,more than20genes were identified that encodemucin-like proteins(18,19)having hallmarksof extensive Thr or Ser stretches suggestive ofglycosylation and signal peptide sequences sug-gesting secretion(table S2).One notable exam-ple is an11,700–amino acid protein with anuninterrupted stretch of308Thr residues(cgd3_720).Although large families of secretedproteins analogous to the Plasmodium multi-gene families were not found,several smallermultigene clusters were observed that encodepredicted secreted proteins,with no detectablesimilarity to proteins from other organisms(Fig.1,A and B).Within this group,at leastfour distinct families appear to have emergedthrough gene expansions specific to the Cryp-R E P O R T S SCIENCE VOL30416APRIL2004443o n O c t o b e r 7 , 2 0 0 9 w w w . s c i e n c e m a g . o r g D o w n l o a d e d f r o mtosporidium clade.These families —SKSR,MEDLE,WYLE,FGLN,and GGC —were named after well-conserved sequence motifs (table S2).Reverse transcription polymerase chain reaction (RT-PCR)expression analysis (20)of one cluster,a locus of seven adjacent CpLSP genes (Fig.1B),shows coexpression during the course of in vitro development (Fig.1C).An additional eight genes were identified that encode proteins having a periodic cysteine structure similar to the Cryptosporidium oocyst wall protein;these eight genes are similarly expressed during the onset of oocyst formation and likely participate in the formation of the coccidian rigid oocyst wall in both Cryptospo-ridium and Toxoplasma (21).Whereas the extracellular proteins described above are of apparent apicomplexan or lineage-specific in-vention,Cryptosporidium possesses many genesencodingsecretedproteinshavinglineage-specific multidomain architectures composed of animal-and bacterial-like extracellular adhe-sive domains (fig.S1).Lineage-specific expansions were ob-served for several proteases (table S2),in-cluding an aspartyl protease (six genes),a subtilisin-like protease,a cryptopain-like cys-teine protease (five genes),and a Plas-modium falcilysin-like (insulin degrading enzyme –like)protease (19genes).Nine of the Cryptosporidium falcilysin genes lack the Zn-chelating “HXXEH ”active site motif and are likely to be catalytically inactive copies that may have been reused for specific protein-protein interactions on the cell sur-face.In contrast to the Plasmodium falcilysin,the Cryptosporidium genes possess signal peptide sequences and are likely trafficked to a secretory pathway.The expansion of this family suggests either that the proteins have distinct cleavage specificities or that their diversity may be related to evasion of a host immune response.Completion of the C.parvum genome se-quence has highlighted the lack of conven-tional drug targets currently pursued for the control and treatment of other parasitic protists.On the basis of molecular and bio-chemical studies and drug screening of other apicomplexans,several putative Cryptospo-ridium metabolic pathways or enzymes have been erroneously proposed to be potential drug targets (22),including the apicoplast and its associated metabolic pathways,the shikimate pathway,the mannitol cycle,the electron transport chain,and HXGPRT.Nonetheless,complete genome sequence analysis identifies a number of classic and novel molecular candidates for drug explora-tion,including numerous plant-like and bacterial-like enzymes (tables S3and S4).Although the C.parvum genome lacks HXGPRT,a potent drug target in other api-complexans,it has only the single pathway dependent on IMPDH to convert AMP to GMP.The bacterial-type IMPDH may be a promising target because it differs substan-tially from that of eukaryotic enzymes (15).Because of the lack of de novo biosynthetic capacity for purines,pyrimidines,and amino acids,C.parvum relies solely on scavenge from the host via a series of transporters,which may be exploited for chemotherapy.C.parvum possesses a bacterial-type thymidine kinase,and the role of this enzyme in pyrim-idine metabolism and its drug target candida-cy should be pursued.The presence of an alternative oxidase,likely targeted to the remnant mitochondrion,gives promise to the study of salicylhydroxamic acid (SHAM),as-cofuranone,and their analogs as inhibitors of energy metabolism in the parasite (23).Cryptosporidium possesses at least 15“plant-like ”enzymes that are either absent in or highly divergent from those typically found in mammals (table S3).Within the glycolytic pathway,the plant-like PPi-PFK has been shown to be a potential target in other parasites including T.gondii ,and PEPCL and PGI ap-pear to be plant-type enzymes in C.parvum .Another example is a trehalose-6-phosphate synthase/phosphatase catalyzing trehalose bio-synthesis from glucose-6-phosphate and uridine diphosphate –glucose.Trehalose may serve as a sugar storage source or may function as an antidesiccant,antioxidant,or protein stability agent in oocysts,playing a role similar to that of mannitol in Eimeria oocysts (24).Orthologs of putative Eimeria mannitol synthesis enzymes were not found.However,two oxidoreductases (table S2)were identified in C.parvum ,one of which belongs to the same families as the plant mannose dehydrogenases (25)and the other to the plant cinnamyl alcohol dehydrogenases.In principle,these enzymes could synthesize protective polyol compounds,and the former enzyme could use host-derived mannose to syn-thesize mannitol.References and Notes1.D.G.Korich et al .,Appl.Environ.Microbiol.56,1423(1990).2.See supportingdata on Science Online.3.M.J.Gardner et al .,Nature 419,498(2002).4.A.T.Bankier et al .,Genome Res.13,1787(2003).5.J.C.Wootton,Comput.Chem.18,269(1994).Fig.1.(A )Schematic showing the chromosomal locations of clusters of potentially secreted proteins.Numbers of adjacent genes are indicated in paren-theses.Arrows indicate direc-tion of clusters containinguni-directional genes (encoded on the same strand);squares indi-cate clusters containingg enes encoded on both strands.Non-paralogous genes are indicated by solid gray squares or direc-tional triangles;SKSR (green triangles),FGLN (red trian-gles),and MEDLE (blue trian-gles)indicate three C.parvum –specific families of paralogous genes predominantly located at telomeres.Insl (yellow tri-angles)indicates an insulinase/falcilysin-like paralogous gene family.Cp LSP (white square)indicates the location of a clus-ter of adjacent large secreted proteins (table S2)that are cotranscriptionally regulated.Identified anchored telomeric repeat sequences are indicated by circles.(B )Schematic show-inga select locus containinga cluster of coexpressed large secreted proteins (Cp LSP).Genes and intergenic regions (regions between identified genes)are drawn to scale at the nucleotide level.The length of the intergenic re-gions is indicated above or be-low the locus.(C )Relative ex-pression levels of CpLSP (red lines)and,as a control,C.parvum Hedgehog-type HINT domain gene (blue line)duringin vitro development,as determined by semiquantitative RT-PCR usingg ene-specific primers correspondingto the seven adjacent g enes within the CpLSP locus as shown in (B).Expression levels from three independent time-course experiments are represented as the ratio of the expression of each gene to that of C.parvum 18S rRNA present in each of the infected samples (20).R E P O R T S16APRIL 2004VOL 304SCIENCE 444 o n O c t o b e r 7, 2009w w w .s c i e n c e m a g .o r g D o w n l o a d e d f r o m。

营养健康甘油二酯油的代谢及生理功能研究进展梅　岚，罗　琪（脂代科技（北京）有限公司，北京 100089）摘 要：油脂过量摄入是导致各项慢性疾病的主要原因。

甘油二酯油因为不同于甘油三酯油的代谢途径而被广泛关注。

在代谢过程中，甘油二酯油不易在机体内累积，而是更多地被氧化而生成能量。

这使得甘油二酯油具有调节血脂水平、抑制体重增长、缓解糖尿病及其并发症等多项有益的生理功能。

本文综述了甘油二酯的代谢途径及生理功能研究进展，以期为甘油二酯油的人体营养学研究提供参考。

关键词：油脂；甘油二酯；脂肪代谢；肥胖Advance in Diacylglycerol Oil for the Metabolism andPhysiological FunctionMEI Lan, LUO Qi(Zhidai Technology (Beijing) Co., Ltd., Beijing 100089, China)Abstract: Excessive intake of fat is the main causes of chronic diseases. Diacylglycerol oil has attracted wide attention because of its different metabolic pathway from triacylglycerol oil. In the process of metabolism, diacylglycerol oil is not easy to accumulate in the body but is oxidized to produce energy. As a result, diacylglycerol oil has many beneficial physiological functions, such as regulating the level of blood lipids, losing weight, inhibiting weight growth, relieving diabetes and its complications. This paper reviews the research progress on the metabolic pathway and physiological function of diacylglycerol in order to provide reference for the study of human nutrition of diacylglycerol.Keywords: oil; diacylglycerol oil; fat metabolism; obesity油脂是日常饮食中重要的营养成分之一，甘油三酯（Triacylglycerol，TAG）是动植物油脂的主要成分，随着居民生活品质的提高，油脂的过量摄入是导致肥胖和一系列慢性疾病的主要原因。