Originally published 28 June 2012; corrected 15 August 2012
https://www.doczj.com/doc/af5507622.html,/cgi/content/full/science.1225829/DC1
Supplementary Materials for
A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive
Bacterial Immunity
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna,*
Emmanuelle Charpentier*
*To whom correspondence should be addressed. E-mail: doudna@https://www.doczj.com/doc/af5507622.html, (J.A.D.);
emmanuelle.charpentier@mims.umu.se (E.C.)
Published 28 June 2012 on Science Express
DOI: 10.1126/science.1225829
This PDF file includes:
Materials and Methods
Figs. S1 to S15
Tables S1 to S3
Full Reference List
Correction: Formatting errors and typos have been corrected. Additionally, the format of the tables has been revised, and a duplicate entry has been removed from table S2.
SUPPLEMENTARY MATERIALS AND METHODS
Bacterial strains and culture conditions. Table S1 lists the bacterial strains used in the study. Streptococcus pyogenes, cultured in THY medium (Todd Hewitt Broth (THB, Bacto, Becton Dickinson) supplemented with 0.2% yeast extract (Oxoid)) or on TSA (trypticase soy agar, BBL, Becton Dickinson) supplemented with 3% sheep blood, was incubated at 37°C in an atmosphere supplemented with 5% CO2 without shaking. Escherichia coli, cultured in Luria-Bertani (LB) medium and agar, was incubated at 37°C with shaking. When required, suitable antibiotics were added to the medium at the following final concentrations: ampicillin, 100 μg/ml for E. coli; chloramphenicol, 33 μg/ml for E. coli; kanamycin, 25 μg/ml for E. coli and 300 μg/ml for S. pyogenes. Bacterial cell growth was monitored periodically by measuring the optical density of culture aliquots at 620 nm using a microplate reader (SLT Spectra Reader). Transformation of bacterial cells. Plasmid DNA transformation into E. coli cells was performed according to a standard heat shock protocol (39). Transformation of S. pyogenes was performed as previously described with some modifications (40). The transformation assay performed to monitor in vivo CRISPR/Cas activity on plasmid maintenance was essentially carried out as described previously (4). Briefly, electro-competent cells of S. pyogenes were equalized to the same cell density and electroporated with 500 ng of plasmid DNA. Every transformation was plated two to three times and the experiment was performed three times independently with different batches of competent cells for statistical analysis. Transformation efficiencies were calculated as CFU (colony forming units) per μg of DNA. Control transformations were performed with sterile water and backbone vector pEC85.
DNA manipulations. DNA manipulations including DNA preparation, amplification, digestion, ligation, purification, agarose gel electrophoresis were performed according to standard techniques (39)with minor modifications. Protospacer plasmids for the in vitro cleavage and S. pyogenes transformation assays were constructed as described previously (4). Additional pUC19-based protospacer plasmids for in vitro cleavage assays were generated by ligating annealed oligonucleotides between digested EcoRI and BamHI sites in pUC19. The GFP gene-containing plasmid has been described previously (41). Kits (Qiagen) were used for DNA purification and plasmid preparation. Plasmid mutagenesis was performed using QuikChange? II XL kit (Stratagene) or QuikChange site-directed mutagenesis kit (Agilent). All plasmids used in this study were sequenced at LGC Genomics or the UC Berkeley DNA Sequencing Facility and are listed in Table S2. VBC-Biotech Services, Sigma-Aldrich and Integrated DNA Technologies supplied the synthetic oligonucleotides and RNAs listed in Table S3.
In vitro transcription and purification of RNA. RNA was in vitro transcribed using T7 Flash in vitro Transcription Kit (Epicentre, Illumina company) and PCR-generated DNA templates carrying a T7 promoter sequence. RNA was gel-purified and quality-checked prior to use. The primers used for the preparation of RNA templates from S. pyogenes SF370, Listeria innocua Clip 11262 and Neisseria meningitidis A Z2491 are listed in Table S3.
Protein purification. The sequence encoding Cas9 (residues 1-1368) was PCR-amplified from the genomic DNA of S. pyogenes SF370 and inserted into a custom pET-based expression vector using ligation-independent cloning (LIC). The resulting fusion construct contained an N-terminal hexahistidine-maltose binding protein (His6-MBP) tag, followed by a peptide sequence containing a tobacco etch virus (TEV) protease cleavage site. The protein was expressed in E. coli strain BL21 Rosetta 2 (DE3) (EMD Biosciences), grown in 2xTY medium at 18°C for 16 h following induction with 0.2 mM IPTG. The protein was purified by a combination of affinity, ion exchange and size exclusion chromatographic steps. Briefly, cells were lysed in 20 mM Tris pH 8.0, 500 mM NaCl, 1 mM TCEP (supplemented with protease inhibitor cocktail (Roche)) in a homogenizer (Avestin). Clarified lysate was bound in batch to Ni-NTA agarose (Qiagen). The resin was washed extensively with 20 mM Tris pH 8.0, 500 mM NaCl and the bound protein was eluted in 20 mM Tris pH 8.0, 250 mM NaCl, 10% glycerol. The His6-MBP affinity tag was removed by cleavage with TEV protease, while the protein was dialyzed overnight against 20 mM HEPES pH 7.5, 150 mM KCl, 1 mM TCEP, 10% glycerol. The cleaved Cas9 protein was separated from the fusion tag by purification on a 5 ml SP Sepharose HiTrap column (GE Life Sciences), eluting with a linear gradient of 100 mM – 1 M KCl. The protein was further purified by size exclusion chromatography on a Superdex 200 16/60 column in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP. Eluted protein was concentrated to ~8 mg.ml-1, flash-frozen in liquid nitrogen and stored at -80°C. Cas9 D10A, H840A and D10A/H840A point mutants were generated using the QuikChange site-directed mutagenesis kit (Agilent) and confirmed by DNA sequencing. The proteins were purified following the same procedure as for the wild-type Cas9 protein.
Cas9 orthologs from Streptococcus thermophilus (LMD-9,YP_820832.1), L. innocua (Clip11262, NP_472073.1), Campylobacter jejuni (subsp. jejuni NCTC 11168, YP_002344900.1) and N. meningitidis (Z2491, YP_002342100.1) were expressed in BL21 Rosetta (DE3) pLysS cells (Novagen) as His6-MBP (N. meningitidis and C. jejuni), His6-Thioredoxin (L. innocua) and His6-GST (S. thermophilus) fusion proteins, and purified essentially as for S. pyogenes Cas9 with the following modifications. Due to large amounts of co-purifying nucleic acids, all four Cas9 proteins were purified by an additional heparin sepharose step prior to gel filtration, eluting the bound protein with a linear gradient of 100 mM – 2 M KCl. This successfully removed nucleic acid contamination from the C. jejuni, N. meningitidis and L. innocua proteins, but failed to remove co-purifying nucleic acids from the S. thermophilus Cas9 preparation. All proteins were concentrated to 1-8 mg.ml-1 in 20 mM HEPES pH 7.5, 150 mM KCl and 1 mM TCEP, flash-frozen in liquid N2 and stored at -80°C.
Plasmid DNA cleavage assay. Synthetic or in vitro-transcribed tracrRNA and crRNA were pre-annealed prior to the reaction by heating to 95°C and slowly cooling down to room temperature.Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) was incubated for 60 min at 37°C with purified Cas9 protein (50-500 nM) and tracrRNA:crRNA duplex (50-500 nM, 1:1) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) with or without 10 mM MgCl2. The reactions were stopped with 5X DNA loading buffer containing 250 mM EDTA, resolved by 0.8 or 1% agarose gel electrophoresis and visualized by ethidium bromide
staining. For the Cas9 mutant cleavage assays, the reactions were stopped with 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) prior to loading on the agarose gel.
Metal-dependent cleavage assay. Protospacer 2 plasmid DNA (5 nM) was incubated for 1 h at 37°C with Cas9 (50 nM) pre-incubated with 50 nM tracrRNA:crRNA-sp2 in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA) supplemented with 1, 5 or 10 mM MgCl2, 1 or 10 mM of MnCl2, CaCl2, ZnCl2, CoCl2, NiSO4 or CuSO4. The reaction was stopped by adding 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA), resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining.
Single-turnover assay. Cas9 (25 nM) was pre-incubated 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with duplexed tracrRNA:crRNA-sp2 (25 nM, 1:1) or both RNAs (25 nM) not pre-annealed and the reaction was started by adding protospacer 2 plasmid DNA (5 nM). The reaction mix was incubated at 37°C. At defined time intervals, samples were withdrawn from the reaction, 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) was added to stop the reaction and the cleavage was monitored by 1% agarose gel electrophoresis and ethidium bromide staining. The same was done for the single turnover kinetics without pre-incubation of Cas9 and RNA, where protospacer 2 plasmid DNA (5 nM) was mixed in cleavage buffer with duplex tracrRNA:crRNA-sp2 (25 nM) or both RNAs (25 nM) not pre-annealed, and the reaction was started by addition of Cas9 (25 nM). Percentage of cleavage was analyzed by densitometry and the average of three independent experiments was plotted against time. The data were fit by nonlinear regression analysis and the cleavage rates (k obs [min-1]) were calculated.
Multiple-turnover assay. Cas9 (1 nM) was pre-incubated for 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mM EDTA) with pre-annealed tracrRNA:crRNA-sp2 (1 nM, 1:1). The reaction was started by addition of protospacer 2 plasmid DNA (5 nM). At defined time intervals, samples were withdrawn and the reaction was stopped by adding 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA). The cleavage reaction was resolved by 1% agarose gel electrophoresis, stained with ethidium bromide and the percentage of cleavage was analyzed by densitometry. The results of four independent experiments were plotted against time (min).
Oligonucleotide DNA cleavage assay. DNA oligonucleotides (10 pmol) were radiolabeled by incubating with 5 units T4 polynucleotide kinase (New England Biolabs) and ~3–6 pmol (~20–40 mCi) [γ-32P]-ATP (Promega) in 1X T4 polynucleotide kinase reaction buffer at 37°C for 30 min, in a 50 μL reaction. After heat inactivation (65°C for 20 min), reactions were purified through an Illustra MicroSpin G-25 column (GE Healthcare) to remove unincorporated label. Duplex substrates (100 nM) were generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95°C for 3 min, followed by slow cooling to room temperature. For cleavage assays, tracrRNA and crRNA were annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) was
pre-incubated with the annealed tracrRNA:crRNA duplex (500 nM) in cleavage assay buffer (20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT, 5% glycerol) in a total volume of 9 μl. Reactions were initiated by the addition of 1 μl target DNA (10 nM) and incubated for 1 h at 37°C. Reactions were quenched by the addition of 20 μl of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products were resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging (Storm, GE Life Sciences). Cleavage assays testing PAM requirements (Fig. 4B) were carried out using DNA duplex substrates that had been pre-annealed and purified on an 8% native acrylamide gel, and subsequently radiolabeled at both 5’ ends. The reactions were set-up and analyzed as above.
Electrophoretic mobility shift assays. Target DNA duplexes were formed by mixing of each strand (10 nmol) in deionized water, heating to 95°C for 3 min and slow cooling to room temperature. All DNAs were purified on 8% native gels containing 1X TBE. DNA bands were visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H2O. Eluted DNA was ethanol precipitated and dissolved in DEPC-treated H2O. DNA samples were 5’ end labeled with [γ-32P]-ATP using T4 polynucleotide kinase (New England Biolabs) for 30 min at 37°C. PNK was heat denatured at 65°C for 20 min, and unincorporated radiolabel was removed using an Illustra MicroSpin G-25 column (GE Healthcare). Binding assays were performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 10% glycerol in a total volume of 10 μl. Cas9 D10A/H840A double mutant was programmed with equimolar amounts of pre-annealed tracrRNA:crRNA duplex and titrated from 100 pM to 1 μM. Radiolabeled DNA was added to a final concentration of 20 pM. Samples were incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing 1X TBE and 5 mM MgCl2. Gels were dried and DNA visualized by phosphorimaging.
In silico analysis of DNA and protein sequences. Vector NTI package (Invitrogen) was used for DNA sequence analysis (Vector NTI) and comparative sequence analysis of proteins (AlignX).
In silico modeling of RNA structure and co-folding. In silico predictions were performed using the Vienna RNA package algorithms (42, 43). RNA secondary structures and co-folding models were predicted with RNAfold and RNAcofold, respectively and visualized with VARNA (44).
SUPPLEMENTARY FIGURES
Fig. S1. The type II RNA-mediated CRISPR/Cas immune pathway. The expression and interference steps are represented in the drawing. The type II CRISPR/Cas loci are composed of an operon of four genes (blue) encoding the proteins Cas9, Cas1, Cas2 and Csn2, a CRISPR array consisting of a leader sequence followed by identical repeats (black rectangles) interspersed with unique genome-targeting spacers (colored diamonds) and a sequence encoding the trans-activating tracrRNA (red). Represented here is the type II CRISPR/Cas locus of S. pyogenes SF370 (Accession number NC_002737) (4). Experimentally confirmed promoters and transcriptional terminator in this locus are indicated (4). The CRISPR array is transcribed as a precursor CRISPR RNA (pre-crRNA) molecule that undergoes a maturation process specific to the type II systems (4). In S. pyogenes SF370, tracrRNA is transcribed as two primary transcripts of 171 and 89 nt in length that have complementarity to each repeat of the pre-crRNA. The first processing event involves pairing of tracrRNA to pre-crRNA, forming a duplex RNA that is recognized and cleaved by the housekeeping endoribonuclease RNase III (orange) in the presence of the Cas9 protein. RNase III-mediated cleavage of the duplex RNA generates a 75-nt processed tracrRNA and a 66-nt intermediate crRNAs consisting of a central region containing a sequence of one spacer, flanked by portions of the repeat sequence. A second processing event, mediated by unknown ribonuclease(s), leads to the formation of mature crRNAs of 39 to 42 nt in length consisting of 5’-terminal spacer-derived guide sequence and repeat-derived 3’-terminal sequence. Following the first and second processing events, mature tracrRNA remains paired to the mature crRNAs and bound to the Cas9 protein. In this ternary complex, the dual tracrRNA:crRNA structure acts as guide RNA that directs the endonuclease Cas9 to the cognate target DNA. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the invading DNA molecule for homology between the protospacer sequence in the target DNA and the spacer-derived sequence in the crRNA. In addition to the DNA protospacer-crRNA spacer complementarity, DNA targeting requires the presence of a short motif (NGG, where N can be any nucleotide) adjacent to the protospacer (protospacer adjacent motif - PAM). Following pairing between the dual-RNA and the protospacer sequence, an R-loop is formed and Cas9 subsequently introduces a double-stranded break (DSB) in the DNA. Cleavage of target DNA by Cas9 requires two catalytic domains in the protein. At a specific site relative to the PAM, the HNH domain cleaves the complementary strand of the DNA while the RuvC-like domain cleaves the noncomplementary strand.
Fig. S2. Purification of Cas9 nucleases. (A) S. pyogenes Cas9 was expressed in E. coli as a fusion protein containing an N-terminal His6-MBP tag and purified by a combination of affinity, ion exchange and size exclusion chromatographic steps. The affinity tag was removed by TEV protease cleavage following the affinity purification step. Shown is a chromatogram of the final size exclusion chromatography step on a Superdex 200 (16/60) column. Cas9 elutes as a single monomeric peak devoid of contaminating nucleic acids, as judged by the ratio of absorbances at 280 and 260 nm. Inset; eluted fractions were resolved by SDS-PAGE on a 10% polyacrylamide gel and stained with SimplyBlue Safe Stain (Invitrogen). (B) SDS-PAGE analysis of purified Cas9 orthologs. Cas9 orthologs were purified as described in Supplementary Materials and Methods. 2.5 μg of each purified Cas9 were analyzed on a 4-20% gradient polyacrylamide gel and stained with SimplyBlue Safe Stain.
Fig. S3. Cas9 guided by dual-tracrRNA:crRNA cleaves protospacer plasmid and oligonucleotide DNA. See Fig. 1.The protospacer 1 sequence originates from S. pyogenes SF370 (M1)SPy_0700, target of S. pyogenes SF370 crRNA-sp1 (4). Here, the protospacer 1 sequence was manipulated by changing the PAM from a nonfunctional sequence (TTG) to a functional one (TGG). The protospacer 4 sequence originates from S. pyogenes MGAS10750 (M4) MGAS10750_Spy1285,target of S. pyogenes SF370 crRNA-sp4 (4). (A) Protospacer 1 plasmid DNA cleavage guided by cognate tracrRNA:crRNA
duplexes. The cleavage products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.M, DNA marker; fragment sizes in base pairs are indicated. (B) Protospacer 1 oligonucleotide DNA cleavage guided by cognate tracrRNA:crRNA-sp1 duplex. The cleavage products were resolved by denaturating polyacrylamide gel electrophoresis and visualized by phosphorimaging.Fragment sizes in nucleotides are indicated. (C) Protospacer 4 oligonucleotide DNA cleavage guided by cognate tracrRNA:crRNA-sp4 duplex. The cleavage products were resolved by denaturating polyacrylamide gel electrophoresis and visualized by phosphorimaging.Fragment sizes in nucleotides are indicated. (A, B, C) Experiments in (A) were performed as in Fig. 1A; in (B) and in (C) as in Fig. 1B. (B, C) A schematic of the tracrRNA:crRNA-target DNA interaction is shown below. The regions of crRNA complementarity to tracrRNA and the protospacer DNA are represented in orange and yellow, respectively. The PAM sequence is indicated in grey.
Fig. S4. Cas9 is a Mg2+-dependent endonuclease with 3’-5’ exonuclease activity. See Fig. 1. (A) Protospacer 2 plasmid DNA was incubated with Cas9 complexed with tracrRNA:crRNA-sp2 in the presence of different concentrations of Mg2+, Mn2+, Ca2+, Zn2+, Co2+, Ni2+ or Cu2+. The cleavage products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.Plasmid forms are indicated.(B) A protospacer 4 oligonucleotide DNA duplex containing a PAM motif was annealed and gel-purified prior to radiolabeling at both 5’ ends. The duplex (10 nM final concentration) was incubated with Cas9 programmed with tracrRNA (nucleotides 23-89) and crRNA-sp4 (500 nM final concentration, 1:1). At indicated time points (min), 10 μl aliquots of the cleavage reaction were quenched with formamide buffer containing 0.025% SDS and 5 mM EDTA, and analyzed by denaturing polyacrylamide gel electrophoresis as in Fig. 1B. Sizes in nucleotides are indicated.
Fig. S5. Dual-tracrRNA:crRNA directed Cas9 cleavage of target DNA is site-specific. See Fig. 1 and fig. S3. (A) Mapping of protospacer 1 plasmid DNA cleavage. Cleavage products from fig. S3A were analyzed by sequencing as in Fig. 1C. Note that the 3’ terminal A overhang (asterisk) is an artifact of the sequencing reaction.(B) Mapping of protospacer 4 oligonucleotide DNA cleavage. Cleavage products from fig. S3C were analyzed by denaturing polyacrylamide gel electrophoresis alongside 5’ end-labeled oligonucleotide size markers derived from the complementary and noncomplementary strands of the protospacer 4 duplex DNA. M, marker; P, cleavage product. Fragment sizes in nucleotides are indicated. (C) Schematic representations of tracrRNA, crRNA-sp1 and protospacer 1 DNA sequences (left) and tracrRNA, crRNA-sp4 and protospacer 4 DNA sequences (right). tracrRNA:crRNA forms a dual-RNA structure directed to complementary protospacer DNA through crRNA-protospacer DNA pairing. The regions of crRNA complementary to tracrRNA and the protospacer DNA are represented in orange and yellow, respectively. The cleavage sites in the complementary and noncomplementary DNA strands mapped in (A) (left) and (B) (right) are represented with red arrows (A and B, complementary strand) and a red thick line (B, noncomplementary strand) above the sequences, respectively.
Fig. S6. Dual-tracrRNA:crRNA directed Cas9 cleavage of target DNA is fast and efficient. See Fig. 1. (A) Single turnover kinetics of Cas9 under different RNA pre-annealing and protein-RNA pre-incubation conditions. Protospacer 2
plasmid DNA was incubated with either Cas9 pre-incubated with pre-annealed tracrRNA:crRNA-sp2 (○), Cas9 not pre-incubated with pre-annealed tracrRNA:crRNA-sp2 (●), Cas9 pre-incubated with not pre-annealed tracrRNA and crRNA-sp2 (□) or Cas9 not pre-incubated with not pre-annealed RNAs (■). The cleavage activity was monitored in a time-dependent manner and analyzed by agarose gel electrophoresis followed by ethidium bromide staining. The average percentage of cleavage from three independent experiments is plotted against the time (min) and fitted with a nonlinear regression. The calculated cleavage rates (k obs) are shown in the table on the right. The results suggest that the binding of Cas9 to the RNAs is not rate-limiting under the conditions tested. Plasmid forms are indicated. The obtained k obs values are comparable to those of restriction endonucleases which are typically of the order of 1-10 min-1 (45-47).
(B) Cas9 is a multiple turnover endonuclease. Cas9 loaded with duplexed tracrRNA:crRNA-sp2 (1 nM, 1:1:1 – indicated with red line on the graph) was incubated with a 5-fold excess of native protospacer 2 plasmid DNA. Cleavage was monitored by withdrawing samples from the reaction at defined time intervals (0 to 120 min) followed by agarose gel electrophoresis analysis (top) and determination of cleavage product amount (nM) (bottom). Standard deviations of three independent experiments are indicated. In the time interval investigated, 1 nM Cas9 was able to cleave ~2.5 nM plasmid DNA.
Fig. S7. Cas9 contains two nuclease domains: HNH (McrA-like) and RuvC-like (N-terminal RNase H fold) resolvase. See Fig. 2. The amino-acid sequence of Cas9 from S. pyogenes SF370 (Accession number NC_002737) is represented.The three predicted RuvC-like motifs and the predicted HNH motif are shaded light blue and purple, respectively. Residues Asp10 and His840, which were substituted by Ala in this study are shown in red and highlighted by a red asterisk above the sequence. Underlined residues are highly conserved among Cas9 proteins from different species. Note that in a previous study and based on computational predictions, Makarova et al. (23) predicted coupling of the two nuclease-like activities, which is now confirmed experimentally in the
present study (Fig. 2 and fig. S8).
Fig. S8. The HNH and RuvC-like domains of Cas9 direct cleavage of the complementary and noncomplementary DNA strand, respectively. See Fig.
2. Protospacer DNA cleavage by cognate tracrRNA:crRNA-directed Cas9 mutants containing mutations in the HNH or RuvC-like domain. (A) Protospacer 1 plasmid DNA cleavage. The experiment was performed as in Fig. 2A. Plasmid DNA conformations and sizes in base pairs are indicated. (B) Protospacer 4 oligonucleotide DNA cleavage. The experiment was performed as in Fig. 2B. Sizes in nucleotides are indicated.
Fig. S9. tracrRNA is required for target DNA recognition. Electrophoretic mobility shift assays were performed using protospacer 4 target DNA duplex and Cas9 (containing nuclease domain inactivating mutations D10A and H840) alone or in the presence of crRNA-sp4, tracrRNA (75nt), or both. The target DNA duplex was radiolabeled at both 5’ ends. Cas9 (D10/H840A) and complexes were titrated from 1 nM to 1 μM. Binding was analyzed by 8% native polyacrylamide gel electrophoresis and visualized by phosphorimaging.
Note that Cas9 alone binds target DNA with moderate affinity. This binding is unaffected by the addition of crRNA, suggesting that this represents sequence nonspecific interaction with the dsDNA. Furthermore, this interaction can be outcompeted by tracrRNA alone in the absence of crRNA. In the presence of both crRNA and tracrRNA, target DNA binding is substantially enhanced and yields a species with distinct electrophoretic mobility, indicative of specific target DNA recognition.
Fig. S10. Minimal region of tracrRNA capable of guiding dual-tracrRNA:crRNA directed cleavage of target DNA. See Fig. 3. A fragment of tracrRNA encompassing a part of the crRNA paired region and a portion of the downstream region is sufficient to direct cleavage of protospacer oligonucleotide DNA by Cas9. (A) Protospacer 1 oligonucleotide DNA cleavage and (B) Protospacer 4 oligonucleotide DNA cleavage by Cas9 guided with a mature cognate crRNA and various tracrRNA fragments. (A, B) Sizes in nucleotides are indicated.
Fig. S11. Dual-tracrRNA:crRNA guided target DNA cleavage by Cas9 is species specific. See Fig. 3. Like Cas9 from S. pyogenes, the closely related Cas9 orthologs from the Gram-positive bacteria L. innocua and S. thermophilus cleave protospacer
DNA when targeted by tracrRNA:crRNA from S. pyogenes. However, under the same conditions, DNA cleavage by the less closely related Cas9 orthologs from the Gram-negative bacteria C. jejuni and N. meningitidis is not observed. Spy, S. pyogenes SF370 (Accession Number NC_002737); Sth, S. thermophilus LMD-9 (STER_1477 Cas9 ortholog; Accession Number NC_008532); Lin, L. innocua Clip11262 (Accession Number NC_003212); Cje, C. jejuni NCTC 11168 (Accession Number NC_002163); Nme, N. meningitidis A Z2491 (Accession Number NC_003116). (A) Cleavage of protospacer plasmid DNA. Protospacer 2 plasmid DNA (300 ng) was subjected to cleavage by different Cas9 orthologs (500 nM) guided by hybrid tracrRNA:crRNA-sp2 duplexes (500 nM, 1:1) from different species. To design the RNA duplexes, we predicted tracrRNA sequences from L. innocua and N. meningitidis based on previously published Northern blot data(4).The dual-hybrid RNA duplexes consist of species-specific tracrRNA and a heterologous crRNA. The heterologous crRNA sequence was engineered to contain S. pyogenes DNA-targeting sp2 sequence at the 5’ end fused to L. innocua or N. meningitidis tracrRNA-binding repeat sequence at the 3’ end.Cas9 orthologs from S. thermophilus and L. innocua, but not from N. meningitidis or C. jejuni, can be guided by S. pyogenes tracrRNA:crRNA-sp2 to cleave protospacer 2 plasmid DNA, albeit with slightly decreased efficiency. Similarly, the hybrid L. innocua tracrRNA:crRNA-sp2 can guide S. pyogenes Cas9 to cleave the target DNA with high efficiency, whereas the hybrid N. meningitidis tracrRNA:crRNA-sp2 triggers only slight DNA cleavage activity by S. pyogenes Cas9. As controls, N. meningitidis and L. innocua Cas9 orthologs cleave protospacer 2 plasmid DNA when guided by the cognate hybrid tracrRNA:crRNA-sp2. Note that as mentioned above, the tracrRNA sequence of N. meningitidis is predicted only and has not yet been confirmed by RNA sequencing. Therefore, the low efficiency of cleavage could be the result of either low activity of the Cas9 orthologs or the use of a nonoptimally designed tracrRNA sequence. (B) Cleavage of protospacer oligonucleotide DNA. 5’-end radioactively labeled complementary strand oligonucleotide (10 nM) pre-annealed with unlabeled noncomplementary strand oligonucleotide (protospacer 1) (10 nM) (left) or 5’-end radioactively labeled noncomplementary strand oligonucleotide (10 nM) pre-annealed with unlabeled complementary strand oligonucleotide (10 nM) (right) (protospacer 1) was subjected to cleavage by various Cas9 orthologs (500 nM) guided by tracrRNA:crRNA-sp1 duplex from S. pyogenes (500 nM, 1:1). Cas9 orthologs from S. thermophilus and L. innocua, but not from N. meningitidis or C. jejuni can be guided by S. pyogenes cognate dual-RNA to cleave the protospacer oligonucleotide DNA, albeit with decreased efficiency. Note that the cleavage site on the complementary DNA strand is identical for all three orthologs. Cleavage of the noncomplementary strand occurs at distinct positions. (C) Amino acid sequence identity of Cas9 orthologs. S. pyogenes, S. thermophilus and L. innocua Cas9 orthologs share high percentage of amino acid identity. In contrast, the C. jejuni and N. meningitidis Cas9 proteins differ in sequence and length (~300-400 amino acids shorter). (D)Co-foldings of engineered species-specific heterologous crRNA sequences with the corresponding tracrRNA orthologs from S. pyogenes (experimentally confirmed, (4)), L. innocua (predicted) or N. meningitidis (predicted). Red, tracrRNA; green, crRNA spacer 2 fragment; grey, crRNA repeat fragment. L. innocua and S. pyogenes hybrid tracrRNA:crRNA-sp2 duplexes share very similar structural characteristics, albeit distinct from the N. meningitidis hybrid tracrRNA:crRNA. Together with the cleavage data described above in (A) and (B),
酪氨酸激酶异常活化在恶性血液病发病 中的作用(1) 】蛋白酪氨酸激酶（protein tyrosine kinase, PTK）在调节细胞生长、活化和分化的信号转导中起着重的作用。基因突变（多半由染色体移位）或者激酶的过度表达可使PTK活力异常增高，并介导异常的信号转导途径，在多种恶性血液病的发生发展中起着主的作用。在慢性骨髓增殖性疾病（CMPD）、急性髓性白血病（AML）和间变性大细胞淋巴瘤的发病中，均存在着PTK的异常活化。进一步研究PTK相关的恶性血液病的发病机理，可以加快特异性的分子靶向治疗的研究进展。 【关键词】酪氨酸激酶恶性血液病；基因突变 Abnormal Activation of Tyrosine Kinases and Its Role in the Pathogenesis of Hematological Malignancies ——Review Abstract Protein tyrosine kinases are key participants in signal transduction pathways that regulate cellular growth, activation and differentiation. Aberrant PTK activity resulting from gene mutation (often accompanying chromosome translocation) or overexpression of these enzymes plays an etiologic role in several clonal hematopoietic malignancies. Other than the causative effect of PTK product of the bcr/abl fusion gene on chronic myelogenous leukemia (CML), more evidence suggests that mutated tyrosine kinases are pivotal in the pathogenesis of most of other chronic myeloproliferative disorders, such as chronic myelomonocytic leukemia (CMML) and hypereosinophilic syndrome (HES). And the exciting results in several dependent groups in 2005 showed that a single nucleotide JAK2 somatic mutation (JAK2V617F mutation) was found to be involved in the pathogenesis of polycythemia vera (PV), essential thrombocythemia (ET) and chronic idiopathic myelofibrosis (CIMF). In the leukogenesis of acute myeloid leukemias (AML), the losing of the control of the proliferation of hematopoietic progenitor cells was principally the results of the aberrant PTK activity, such as FLT3 and C kit overexpression. It works together with the loss of function mutation genes in promoting progenitor cell differentiation to confer AML's phenotypes. These upregulated PTK molecules represent attractive disease specific
实验名称：基因克隆 实验器材:荧光定量PCR仪、摇床、离心机、生工PCR产物纯化试剂盒、恒温加热器、 NEB连接体系、灭菌纯水、JM109感受态、冰、LB培养基、酒精灯、涂棒、氨苄、氨苄抗性平板、甘油等； 操作步骤： 1、可通过PCR进行拼接获得目的基因的，过柱纯化（生工试剂盒根据说明书进行纯化， 在最后一步的洗脱可以用预热的灭菌纯水洗脱，在加灭菌纯水洗脱的时候一定要加在纯化柱子的膜中间）； 2、选择合适的载体（EZ-T）用连接酶进行连接，NEB体系，16℃过夜连接 T4lages 1.0 10×T4buffer 2.0 EZ-T 1.0 目的基因8.0 DdH2O 8.0 ＿＿＿＿＿＿＿＿＿ 20ul 3、取100μl摇匀后的JM109感受态细胞悬浮液（如是冷冻保存液，则需化冻后马上进行下 面的操作），加入10μl连接产物，轻轻摇匀，冰上放置30min后，于42度水浴中保温90s，然后迅速在冰上冷却2min； 4、加入500μl LB液体培养基，混匀于37℃振荡培养45min使受体菌恢复正常生长状态并 使转化体产生抗药性； 5、将恢复培养的菌体5000rpm离心3min，移去上层LB培养基，用余下的200μl重悬菌体， 并用灭菌玻璃推子(酒精灯上烧后冷却)，均匀涂布于琼脂凝胶表面（氨苄抗性），37℃倒置培养12~16小时； 6、挑取多个单克隆菌落分别接种到1ml含有抗生素（氨苄）的LB液体培养基中，37℃振 荡培养3h； 7、培养1-2小时即可以利用PCR（定量或定性）进行鉴定； 8、选取初步鉴定阳性的菌液送测序，测序正确后甘油保存（甘油的浓度为30%-50%），充 分混匀，-80℃保存；
毕业设计/论文 开题报告 课题名称红豆杉中MYB家族基因克隆及表达分析类别毕业论文 系别城市建设学院 专业班生物工程0701班 姓名于凯 评分 指导教师 华中科技大学武昌分校
华中科技大学武昌分校学生毕业论文开题报告
癌活性，对于治疗卵巢癌、乳腺癌等疗效突出。但是由于含量少、提取困难等诸多因素，高纯度紫杉醇价格昂贵，每公斤200万元人民币左右。因此，近年来国内外许研究人员、实验室和公司一直试图通过生物合成、化学合成、微生物提取、组织和细胞培养、寻找类似物等途径来解决紫杉醇的药源短缺问题。 研究紫杉醇的生物合成，尤其一些限速反应步骤机理的阐明对于人为定向的提高合成效率，克隆重组形成关键酶基因从而提高紫杉醇的产量意义重大。从理论上来说这是一个好方法，但是紫杉醇的合成途径非常复杂，涉及到多种酶以及很多分支途径，单纯依靠转化一、两种限速酶基因，只能保证转入的限速酶表达量提高，使之不再是限速因素，但其它阶段对于最终产量的限制依然存在，而且同时转入多种基因的可行性非常低，这种方法的缺陷很明显。 若采用化学合成，如从红豆杉植物中分离得到的巴卡亭Ⅲ经过四步化学过程可合成紫杉醇，为合成紫杉醇提供了新途径[5]。但化学合成从实质意义上说还没有取得彻底的突破，目前还不具备应用价值。 如果从共生真菌中直接提取紫杉醇，能够利用真菌生长速度快的优势，但目前分离的菌株无论从种类还是数量上都远不够工业化的要求，而且还存在很多不确定因素[1]。生产紫杉醇的微生物大多是与红豆杉共生的真菌，其紫杉醇含量极微，并且这些真菌的培养和大规模发酵困难，菌株衰退也是一个难题。 另外，红豆杉愈伤组织和细胞培养生产紫杉醇是研究的热点之一，是工厂化大规模生产紫杉醇的重要手段之一。但运用植物组织、细胞培养技术生产紫杉醇仍处在实验室阶段，如何获得高含量、产紫杉醇稳定的愈伤组织一直都是组织培养、细胞培养生产紫杉醇的关键。 1.1.3关于MYB基因 ①MYB基因 目前，在几乎所有的真核生物中都发现了与禽类逆转录病毒癌基因和细胞原癌基因c-MYB相似的基因，它们的编码产物在结构和功能上具有高度保守的DNA结合域，是一类转录因子[6]。在植物中首先从玉米中克隆了含有MYB结构域的转录因子C1基因，之后在植物中发现的MYB相关基因的数量迅速增加[7]。
植物基因的克隆 08医用二班姚桂鹏0807508245 简介 克隆（clone）是指一个细胞或一个生物个体无性繁殖所产生的后代群体。通常所说的基因克隆是指基于大肠埃希菌的DNA片段（或基因）的扩增，主要过程包括目标DNA的获取、重组载体的构建、受体细胞的转化以及重组细胞的筛选和繁殖等。本文主要介绍植物基因的特点、基因克隆的载体、基因克隆的工具酶、基因克隆的策略以及植物目的基因的分离克隆方法等内容。 关键词 植物基因基因克隆载体工具酶克隆策略分离克隆方法 Plant gene cloning Introduction Cloning (clone) refers to a cell or an individual organisms asexual reproduction produced offspring. Usually said cloning genes means
based on escherichia coli segment of DNA (or genes), including the main course target DNA, restructuring of the carrier, transformation of receptor cells and reorganization of screening and reproductive cells. This paper mainly introduces the characteristics of plant gene and gene cloning and carrier, gene clone tool enzyme, gene cloning and plant gene strategy of separation cloning method, etc. Keywords Plant gene cloning tool enzyme gene cloning vector method of separation of cloning strategy 一、植物基因的结构和功能 基因（gene）是核酸分子中包含了遗传信息的遗传单位。一般来说，植物基因都可分为转录区和非转录的调控区两部分。 （一）植物基因的启动子 启动子（promoter）是指在位于结构基因上游决定基因转录起始的区域，植物积阴德启动子包括三个较重要的区域，一时转录起始位点，而是转录起始位点上游25~40bp的区域，三是转录起始位点上游-75bp处或更远些的区域。 （二）植物基因的增强子序列
一、组织总RNA的提取 相关试剂：T rizol；氯仿；苯酚；异丙醇；75%乙醇；RNase-free水 相关仪器：制冰机；液氮&研钵/生物样品研磨仪；高速离心机；移液器（1ml、200μl、100μl/50μl）；涡旋振荡仪；恒温金属浴。 相关耗材：解剖工具，冰盒，离心管，离心管架，吸头（1ml，200μl/300μl），一次性手套，实验手套。 实验步骤 1.取暂养草鱼，冰上放置一段时间，然后解剖，剪取肠道50~100mg，放入研钵中，加入 液氮迅速研磨，然后加入1ml 预冷TRIzol试剂，充分研磨至无颗粒物存在。 2.转移到离心管中，室温放置5min，使细胞充分裂解； 3.按1ml Trizol加入200μl氯仿，盖上盖子，迅速充分摇匀15s，然后室温放置3min； 4.4℃，,12000g 离心15min； 此时混合物分为三层，下层红色的苯酚氯仿层，中间层和上层无色水相；RNA存在于无色水相中； 5.小心吸取上清液，千万不要吸取中间界面，否则有DNA污染；转移至一个新的离心管， 加入等体积的异丙醇，轻轻混匀； 6.室温放置10min；4℃，,12000g 离心10min； 7.弃上清，加入1ml 75%乙醇洗涤；涡旋，悬浮沉淀；4℃，,12000g 离心5min； 8.弃上清；可以再次用75%乙醇洗涤沉淀； 9.弃上清；用移液器轻轻吸取管壁或管底的残余乙醇，注意不要吸取沉淀；室温放置5min 晾干沉淀；（RNA样品不要过于干燥，否则极难溶解） 10.沉淀中加入30μl RNase-free水，轻弹管壁，使RNA溶解。 RNA质量检测 相关试剂：溴酚蓝，TEB/TAE电泳缓冲液，溴乙锭（EB） 相关仪器：（超微量分光光度计，移液器（2.5μl 或2μl 规格，10μl规格），电子天平，电泳仪，电泳槽，凝胶成像仪，微波炉，制冰机） 相关耗材：（无菌无绒纸，吸头，离心管架，PCR管，PCR管架，锥形瓶，烧杯，一次性手套，实验手套，冰盒） （1）RNA纯度的检测：测定其OD260和OD280的值，根据其OD260/ OD280的比值，当其比值在1.9~2.1之间，说明提取的总RNA纯度比较高，没有蛋白质和基因组的污染。 （2）RNA完整性的检测：取2μlRNA，与2μl溴酚蓝混匀，用1%的琼脂糖进行凝胶电泳，20min后，在凝胶成像系统中观察效果。当28S与18S条带清晰，且亮度比大约是2:1时，5S条带若隐若现，而且没有其它条带时，说明完整性不错，可以用于下游逆转录实验。
细胞色素C氧化酶亚基Ⅱ可溶性结构域的结构改变、转换和功能 研究 细胞色素 c 氧化酶是哺乳动物线粒体或细菌呼吸链上的末端酶，催化电子从细胞色素 c 到 O2 的转移并使后者还原为水。该酶是一个复合的膜蛋白，包括多个亚基及不同的金属活性中心。 其中，亚基II中的 CuA 中心被认为是电子从细胞色素 c 进入该酶的入口，在生理过程中起着重要作用。细胞色素 c 氧化酶的缺损或不足可导致许多疾病，在一些退行性疾病 (如：老年痴呆症) 或癌症研究中已发现了与其相关的基因突变。 本工作以该酶的亚基 II 可溶区为研究对象，成功表达并获得了Paracoccus versutus 的细胞色素 c 氧化酶亚基II可溶区重组蛋白 (128-280位的氨基酸序列)；通过基因工程方法构建突变体蛋白，研究了 Trp121 和 Val162氨基酸残基在该酶中的重要地位，报道了突变和其它物理化学因素引起的蛋白结构改变、转换，以及与此相关的性质和功能变化。主要工作和结果如下：(一) 细胞色素 c 氧化酶亚基II可溶区蛋白的体外表达、纯化及性质表征以大肠杆菌BL21 (DE3) 为表达载体，建立并优化了表达条件，获得了细菌Paracoccus versutus 的细胞色素 c 氧化酶亚基II可溶区重组蛋白。 该蛋白大量存在于包涵体中，通过体外复性，金属中心重组和FPLC纯化，获得了水溶性的紫色蛋白，在SDS-PAGE中表现为单一条带。经电喷雾质谱测定分子量，蛋白的成功制备得到了进一步确证。 采用紫外-可见和园二色谱表征了蛋白的金属活性中心和二级结构。紫外可见光谱显示出明显的 CuA 特征，360，480，530 和 810 nm 的吸收带是双核混
苏明慧，汪一荣，杨艳梅，等．细胞色素Ｃ氧化酶亚基Ｃｏｘ４的克隆及高效表达［Ｊ］．江苏农业科学，２０１８，４６（１５）：３１－３４．ｄｏｉ：１０．１５８８９／ｊ．ｉｓｓｎ．１００２－１３０２．２０１８．１５．００８ 细胞色素Ｃ氧化酶亚基Ｃｏｘ４的克隆及高效表达 苏明慧，汪一荣，杨艳梅，商巾杰 （南京师范大学生命科学学院，江苏南京２１００４６） 摘要：Ｃｏｘ４是电子传递链上复合体Ⅳ的１个关键亚基，实现Ｃｏｘ４的高效表达以及制备其抗体，对于线粒体功能研究至关重要。根据裂殖酵母序列信息数据库（Ｓ．ｐｏｍｂｅ＿ＧｅｎｅＤＢ）中ｃｏｘ４的基因序列（登录号：ＳＰＡＣ１２９６．０２）设计引物，以粟酒裂殖酵母的ｃＤＮＡ序列为模板，通过ＰＣＲ扩增技术得到目标蛋白的基因，然后通过克隆构建到含有Ｔ７强启动子的表达载体ｐＥＴ－２８ａ（＋）上，并将其转入大肠杆菌ＢＬ２１（ＥｓｃｈｅｒｉｃｈｉａｃｏｌｉＢＬ２１）中实现重组工程菌株的构建。结果表明，通过一系列条件优化实现了对重组工程菌株中Ｃｏｘ４的高效表达，并纯化得到大量相应蛋白，最后以纯化的蛋白为抗原成功制备了Ｃｏｘ４抗体。Ｃｏｘ４抗体的成功制备为粟酒裂殖酵母中线粒体功能研究奠定了基础。 关键词：线粒体；粟酒裂殖酵母；细胞色素Ｃ氧化酶；Ｃｏｘ４ 中图分类号：Ｑ７８５ 文献标志码：Ａ 文章编号：１００２－１３０２（２０１８）１５－００３１－０３ 收稿日期：２０１７－０３－１５ 基金项目：国家自然科学基金（编号：３１４０００３２）；江苏省高校自然科学研究项目（编号： １３ＫＪＢ１８００１０）。作者简介：苏明慧（１９９１—），女，河南周口人，硕士研究生，从事粟酒裂殖酵母线粒体蛋白结构与功能研究。Ｅ－ｍａｉｌ：１５７９２５７４８４＠ｑｑ．ｃｏｍ 。通信作者：商巾杰，博士，副教授，从事裂殖酵母中与人类疾病相关基因的结构和功能研究。Ｅ－ｍａｉｌ：ｓｈａｎｇｊｉｎｊｉｅｙ＠１６３．ｃｏｍ。 线粒体有自己的基因组，是一种半自主性的细胞器［１－２］ ，几乎存在于所有真核生物中。线粒体能够进行氧化磷酸化和 三磷酸腺苷（ ＡＴＰ）的合成，是有氧呼吸的主要场所［３－４］ ，可为细胞的生命活动提供能量，被称为细胞内的发动机［５］ 。同 时，线粒体还参与调控细胞的信息传递、细胞的凋亡和细胞的分化等一系列生理过程，并且还对细胞生长和细胞周期进行 调控［ ６］，因此，线粒体对细胞的生长至关重要［５］ 。氧化磷酸化是一个电子传递过程，依赖于线粒体的电子传递链。线粒 体的电子传递链对于线粒体功能的正常发挥十分重要［７］ ，一旦电子传递链受损会引起很多疾病的发生［８－９］。在粟酒裂殖 酵母中，电子传递链由４种复合体组成，而组成这些复合体的蛋白是由核基因组和线粒体基因组共同编码的。细胞色素Ｃ氧化酶（复合物Ⅳ）是线粒体中电子传递链的末端酶，催化电 子从细胞色素Ｃ转移到氧［１０］，核编码的细胞色素Ｃ氧化酶亚 基４（Ｃｏｘ４）是其中１个关键亚基。由此可见，在对线粒体功能进行研究时，不可避免地需要对线粒体中构成这些复合体的蛋白水平进行检测，因此制备这些蛋白的相应抗体就显得尤为重要。本研究主要是将粟酒裂殖酵母中构成复合体Ⅳ的细胞色素Ｃ氧化酶亚基Ｃｏｘ４在大肠杆菌中进行高效表达，并制备相应的抗体。１ 材料与方法１．１ 试验材料 １．１．１ 菌株与质粒 粟酒裂殖酵母单倍体菌株ｙＨＬ６３８１［ｈ＋，组氨酸生物合成缺陷型（ｈｉｓ３－Ｄ１），亮氨酸生物合成缺 陷型（ｌｅｕ１－３２），尿嘧啶生物合成缺陷型（ｕｒａ４－Ｄ１８），腺嘌呤生物合成缺陷型（ ａｄｅ６－Ｍ２１０）］，由南京师范大学微生物所实验室保存。质粒ｐ ＥＴ－２８ａ（＋）为笔者所在实验室保存。１．１．２ 试剂 ｒＴａｑＤＮＡ聚合酶、限制性内切酶、ＳｏｌｕｔｉｏｎⅠ（货号为６０２２）、蛋白ｍａｒｋｅｒ，购自ＴａＫａＲａ公司；ＤＮＡｍａｒｋｅｒ，购自北京全式金生物技术有限公司；ＤＮＡ割胶回收试剂盒、ＰＣＲ过柱纯化试剂盒，购自北京博大泰克生物基因技术有限责任公司；十二烷基硫酸钠（ｓｏｄｉｕｍｄｏｄｅｃｙｌｓｕｌｆａｔｅ，简称ＳＤＳ）、琼脂粉、４－羟乙基哌嗪乙磺酸（ＨＥＰＥＳ）、山梨醇（ｓｏｒｂｉｔｏｌ）、三羟甲基氨基甲烷（Ｔｒｉｓ）、３－（Ｎ－吗啉基）丙磺酸（ ＭＯＰＳ）、３０％丙烯酰胺和乙二胺四乙酸（ＥＤＴＡ），购自索莱宝公司；酵母粉，购自ＯＸＯＩＤ公司；腺嘌呤、尿嘧啶、精氨酸、组氨酸、卡那霉素（Ｋａｎ），购自Ｓｉｇｍａ公司；ＲＮＡ提取试剂盒，ＯＭＥＧＡ（货号为Ｒ６８７０－００），购自南京贝纳生物技术有限公司；ｉＳｃｒｉｐｔｃＤＮＡ合成试剂盒（货号为１７０８８９０），购自南京润亚生物科技发展有限公司； ＰＣＲ引物，由南京思普金生物工程技术服务有限公司合成；常用试剂为分析纯级，购自国药集团化学试剂有限公司和生工生物工程（上海）股份有限公司。 １．１．３ 培养基 （１）ＬＢ培养基配方（１００ｍＬ）：１ｇ胰蛋白胨， ０．５ｇ酵母粉，１ｇ氯化钠，在固体培养基中添加２ｇ琼脂粉。（２）ＬＢ＋Ｋａｎ培养基：在ＬＢ培养基中添加卡那霉素至终浓度为５０ｍｇ／Ｌ。（３）ＹＥＳ培养基配方（１００ｍＬ）：３．０ｇ葡萄糖，０．５ｇ酵母粉，２２．５０ｍｇ腺嘌呤，２２．５０ｍｇ尿嘧啶，２２．５０ｍｇ亮氨酸，２２．５０ｍｇ组氨酸。１．２ 试验方法 １．２．１ 引物的设计 根据裂殖酵母序列信息数据库（Ｓ．ｐｏｍｂｅ＿ＧｅｎｅＤＢ）中登录号为ＳＰＡＣ１２９６．０２的基因序列，通过ｈｔｔｐｓ：／／ｉｈｇ．ｇｓｆ．ｄｅ／ｉｈｇ／ｍｉｔｏｐｒｏｔ．ｈｔｍｌ预测线粒体定位序列，将线粒体定位序列去掉后，设计上下游特异性引物，在正向引物的５′端加入ＮｄｅⅠ酶切位点，在互补链引物的５′端加入ＸｈｏⅠ酶切位点（下划线分别表示ＮｄｅⅠ、ＸｈｏⅠ酶切位点）：正向引物：５′－ＧＧＡＡＴＴＣＣＡＴＡＴＧＡＡＴＧＡＧＣＡＡＡＡＣＧＴＴＧＴＡＡＡＡＧＣＣ－ — １３—江苏农业科学 ２０１８年第４６卷第１５期
3 结果与分析 3.1质粒提取 用醋酸铵法提取pET-28a 和pEGFP-N3质粒后，进行琼脂糖电泳检测质粒是否提取成功。得到电泳结果，如图一所示，3、4号泳道有明显清晰的条带说明pEGFP-N3提取成功。1、2泳道同样有明显清晰的条带，说明pET-28a 提取成功。 3.2 双酶切 用BamH1和Not1分别对pEGFP-N3和pET-28a 双酶切。1、2号泳道为pEGFP-N3的酶切结果，如图二所示，电泳会得到两条带，说明pEGFP-N3酶切成功。4号泳道为pET-28a 的酶切产物的电泳有明显条带，证明酶切成功。 3.3 抗性筛选 通过氯化钙法制备DH5α感受态细胞，用热激发将pET-28a-GFP 转入DH5α感 图 1 pET-28a 和pEGFP-N3质粒提取电泳图 1、2泳道为pET-28a 电泳结果 3、4号泳道为pEGFP-N3电泳结果 图 2 BamH1、Not1双酶切 pEGFP-N3和pET-28a 1、2号泳道为pEGFP-N3酶切产物 3号泳道为pEGFP-N3原始质粒 4号泳道为pET-28a 酶切产物 5号用泳道为pET-28a 原使质粒
受态细胞。转化重组质粒后涂平板，进行重组质粒的抗性筛选。因为28a中含有 抗卡那基因，所以筛选后可以得到含28a的重组质粒。从图中可以看出1号平板 长出较多菌落，说明DH5α感受态细胞存活。2号平板无菌落生长，说明DH5α中 不含抗卡那基因。3号板生长出较少菌落，证明卡那有活性。4号板无菌落生长。 失败原因其一可能是在倒了第一个平板加入卡那后，由于倒平板速度太慢，导致 培养基凝固，影响了卡那的浓度和活性。其二可能是在转化过程中，离心后，弃 上清的过程中，将沉淀和上清混在了一起，影响了溶液的浓度。 图3重组质粒转化DH5α感受态细胞 1号图为不含卡那的阴性对照 2号图为含卡那的阴性对照 3号图为含卡那的自提pET-28a的阳性对照 4号图为含卡那的连接产物结果 3.4PCR鉴定 经PCR扩增后，进行琼脂糖凝胶电泳检测是否扩增成功，得到电泳结果如图 四所示，结果表明，1、2泳道的条带约为700bp，说明成功扩增出含有GFP的基 因。DNA电泳检验扩增片段,选出能够得到700bp左右片段的阳性克隆。 图4阳性重组菌的PCR鉴定 1、2号泳道为重组质粒转化结果
蛋白酪氨酸激酶综述 目前至少已有近六十种分属20个家族的受体酪氨酸激酶被子识别。所有受体酷氨酸激酶都属于I型膜蛋白，其分子具有相似的拓朴结构：糖基化的胞外配体结合区，疏水的单次跨膜区，以及胞内的酪氨酸激酶催化结构域及调控序列。不同受体酪氨酸激酶结合，将导致受体发生三聚化，并进一步使受体胞内区特异的受体酪氨酸残基发生自身磷酸化或交叉磷酸化，从而激活下游的信号转导通路。许多肿瘤的发生、发展都与酪氨酸激酶的异常表达有着极其密切的联系，下面将对几类与肿瘤的发生发展最为密切的受体酪氨酸激酶的研究迸展做一简介。 一、表皮生长因子受体（Epidermal grovth factor receptor, EGFR）家族 EGFRPE包括EGFR、ErbB2、ErbB4等4个成员，其家族受体酪氨酸激酶（RTK）以 单体形式存在，在结构上由胞外区、跨膜区、胞内区3个部分组成，胞外区具有2个半氨酸丰富区，胞内区有典型的ATP结合位点和酪氨酸激酶区，其酪氨酸激酶活性在调节细胞增殖及分化中起着至关重要的作用。 人的egfr基因定位于第7号染色体的短臂（7p12.3-p12.1），它编码的产物EGFR由1210个氨基酸组成，蛋白分子量约为170kDa，其中，712-979位属于酪氨酸激酶区。EGFR的专一配体有EGF、TGF、amphiregulin,与其他EGFR家庭成员共有的配体有（cellulin(BTC)、heparin-binding EGF(HB-EGF)、Epiregulin(EPR) ）等。 EGFR在许多上皮业源的肿瘤细胞中表达，如非小细胞性肺癌，乳腺癌、头颈癌，膀胱癌，胃癌，前列腺癌，卵巢癌、胶质细胞瘤等。另外，在一些肿瘤如恶性胶质瘤、非小细胞性肺癌、乳腺癌、儿童胶质瘤、成神经管细胞瘤及卵巢癌等中还可检测到EGFR缺失。最为常见的EGFR缺失突变型是EGFRⅧ，EGFR Ⅷ失去了配体结合区，但是可自身活化酪氨酸激酶，刺激下游信号通路的激活，而不依赖于与其配全结合。EGFR在许多肿瘤中的过表达和/或突变，借助信号转导至细胞生长失控和恶性化。另外，EGFR的异常表达还与新生血管生成，肿瘤的侵袭和转移，肿瘤的化疗抗性及预后密切相关。EGFR高表达的肿瘤患者，肿瘤恶性程度高，易发生转移，复发间期短，复发率高，患者的存活期短。 ErbB2，又名HER-2/neu，是EGFR家族的第二号成员，ErbB2通过与EGFR家族中其它三位成员构成异源二聚体，而发挥生物学作用，尚未发现能与其直接结合的配体。编码ErbB2的基因neu最早从大鼠神经母细胞瘤中分离得到，人类体细胞内neu基因的同源基因，又称为HER-2或erbB2，位于人第17号染色体的长臂（17q21.1），它编码的产物ErbB2由1255个氨基酸组成，蛋白分子量约为185Kda，其中，720-987位属于酪氨酸激酶区。 ErbB2通常只在胎儿时期表达，成年以后只在极少数组织内低水平表达。然而在多种人类肿瘤中却过度表达，如乳腺癌（25-30%）、卵巢癌（25-32%、肺静癌（30-35%）、原发性肾细胞癌（30-40%）等。过度表达的原因主要是ErbB2基因扩增（95%）或转录增多（5%）。 1987年，Slamon等人首行先报道了ErbB2扩增和乳腺癌临床预后不良之间的显著关系，其显著性高于雌激素、孕激素等指标，并在以后的研究中得到大量证实。随后，ErbB2表达水平和乳腺癌治疗效果间的关系得到广泛研究，人们发现ErbB2高表达乳腺癌患者对他莫昔芬（tamoxifen）治疗、单独的激素疗法、以及环磷酰胺、甲氨喋呤、5-氟脲嘧啶联合化疗产生耐受。研究还表明，ErbB2在细胞的恶性转化中发挥重要作用，并能促进恶性肿瘤转移。ErbB2受体过度表达往往提示乳腺癌恶性程度高，转移潜力强，进展迅速，化疗缓解期短，易产生化疗和激素治疗抗性，生存率和生存期短，复发率高。 和ErbB4对肿瘤的作用目前尚不清楚，但在肿瘤形成模型的临床前研究发现，ErbB3、Erb3与EGFR、ErbB2共表达后会使肿瘤恶性程度明显增加。 二、血管内皮细胞生长因子受体（Vascular endothelial growth factor receptor, VEGFR）家族VEGFR家族的成员包括：VEGFR1（Flt-1）、VEGFR2（KDR/Flk-1）、VEGFR3（Flt-4）,这一家族的受体在细胞外存在着7个免疫球蛋白样的结构域，在胞内酪氨酸激酶区则含有一段亲水手插入序列。
蛋白酪氨酸激酶简介 癌症极大威胁人类健康，抗肿瘤研究是当今生命科学中极富挑战性且意义重大的领域。目前，临床上常用的抗肿瘤药物主要是细胞毒类药物，这类抗癌药具有难以避免的选择性差、毒副作用强、易产生耐药等缺点。近年来，随着生命科学研究的飞速进展，恶性肿瘤细胞内的信号转导、细胞周期的调、细胞凋亡的诱导、血管生成以及细胞与胞外基质的相互作用等各种基本过程正在被逐步阐明。以一些与肿瘤细胞分化增殖相关的细胞信号转导通路的关键酶作为药物筛选靶点，发现选择性作用于特定靶点的高效、低毒、特异性强的新型抗癌药物已成为当今抗肿瘤药物研究开发的重要方向。 蛋白酪氨酸激酶是一类具有酪氨酸激酶活性的蛋白质，可分为受体型和非受体型两种，它们能催化ATP上的磷酸基转移到许多重要蛋白质的酪氨酸残基上，使其发生磷酸化。蛋白酪氨酸激酶在细胞内的信号转导通路中占据了十分重要的地位，调节着细胞体内生长、分化、死亡等一系列生理化过程。 蛋白酪氨酸激酶功能的失调则会引发生物体内的一系列疾病。已有的资料表明，超过50%的原癌基因和癌基因产物都具有蛋白酪氨酸激酶活性，它们的异常表达将导致细胞增殖调节发生紊乱，进而导致肿瘤发生。此外，酪氨酸基酶的异常表达还与肿瘤的侵袭和转移，肿瘤新生血管的生成，肿瘤的化疗抗性密切相关。因此，以酪氨酸激酶为靶点进行药物研发成为国际上抗肿瘤药物研究的热点，为此投入的研究经费也是其它任何一个非传统的肿瘤靶点所无法匹敌的。 目前为止，已有十多种蛋白酪氨酸激酶抑制剂和抗体进入I-Ⅱ期临床试验阶段，个别的已经上市，并取得了令人鼓舞的治疗结果。基中，Genentech公司和罗氏药厂联合研究和生产的HerceptinTM(Trastuzumab)是一种抗酪氨酸激酶受体HER2/neu的人源化的单克隆抗体。1998年，美国食品的药物管理局（Food and Drug Administration, FDA）正式批准Herceptin用于治疗某些HER2阳性的转移性乳腺癌。2001年5月，N ovartis公司研发的针对酪氨酸激酶Bcr-Abl的抑制剂GleevecTM (imatinib mesylate)由于对治疗慢性髓样白血病（chronic myelogenous leukemia,CML）具有非常好的疗效，尚未完成Ⅲ期临床就被FDA批准提前上市，用于治疗费城染色体呈阳性（Philadelphia chromosome – positive, Ph+）的慢性髓样白血病患者，引起了巨大的轰动。GleevecTM是第一个在了解癌症的病因后鸽是设计开发，并取得了显著成效和的肿瘤治疗药物，它的研发成功可以说是癌症治疗的一个里程碑。这一重大成就被美国《科学》杂志列入2001年度十大科技新闻。纽约《时代》杂志将其作为杂志的封面，称GleevecTM 开创了药物研发的新时代。2002年2月，美国FDA又批准GleevecTM 用于胃肠基质瘤（gastrointestinal stromal tumors, GLST）的治疗。2002年7月，AstraZeneca公司研发的IressaTM (ZD1839又被美国FDA批准用于治疗经过标准含铂类方案和紫杉萜化疗后仍然继续恶化的终未期非小细胞肺癌患者，这也是第一种用于实体瘤治疗的针对特定靶点挑战分子酪氨酸激酶抑制剂。Herceptin，Gleevec以及Iressa的上市进一步证明了以特定靶点尤其是以酪氨酸激酶为靶点进行抗肿瘤药物的研发是21世纪最有可能获得突破性进展的抗肿瘤药物领域，具有十分广阔的前景。
拟南芥基因克隆的策略与途径 拟南芥（Arabidopsis thaliana）是一种模式植物，具有基因组小（125 Mbp）、生长周期短等特点，而且基因组测序 已经完成（The Arabidopsis Genomic Initiative, 2000）。同时，拟南芥属十字花科（Cruciferae），具有高等植物 的一般特点，拟南芥研究中所取得成果很容易用于其它高等植物包括农作物的研究，产生重大的经济效益，特别是十字 花科中还有许多重要的经济作物，与人类的生产生活密切相关，因此目前拟南芥的研究越来越多地受到国际植物学及各 国政府的重视。 基因(gene)是遗传物质的最基本单位,也是所有生命活动的基础。不论要揭示某个基因的功能,还是要改变某个基因的功 能,都必须首先将所要研究的基因克隆出来。特定基因的克隆是整个基因工程或分子生物学的起点。本文就基因克隆的 几种常用方法介绍如下。 1、图位克隆 Map-based cloning, also known as positional cloning, first proposed by Alan Coulson of the University of Cambridge in 1986, Gene isolated by this method is based on functional genes in the genome has a relatively stable loci, in the use of genetic linkage analysis or chromosomal abnormalities of separate groups will queue into the chromosome of a specific location, By constructing high-density molecular linkage map, to find molecular markers tightly linked with the aimed gene, continued to narrow the candidate region and then clone the gene and to clarify its function and biochemical mechanisms. 图位克隆（map-based clonig）又称定位克隆（positoinal cloning），1986年首先由剑桥大学的Alan Coulson提出。用该方法分离基因是根据功能基因在基因组中都有相对较稳定的基因座，在利用分离群体的遗传连锁分析或染色体异常将基因伫到染色体的1个具体位置的基础上，通过构建高密度的分子连锁图，找到与目的基因紧密连锁的分子标记，不断缩小候选区域进而克隆该基因，并阐明其功能和生化机制。 用该方法分离基因是根据目的基因在染色体上的位置进行的，无需预先知道基因的DNA序列，也无需预先知道其表达产物的有关信息。它是通过分析突变位点与已知分子标记的连锁关系来确定突变表型的遗传基础。近几年来随着拟南芥基因组测序工作的完成，各种分子标记的日趋丰富和各种数据库的完善，在拟南芥中克隆一个基因所需要的努力已经大大减少了（图1）。
基因克隆及转基因 一、基因克隆及转基因过程 1、设计引物 软件是，用到里面的PrimerSelect和EditSeq。 一般原则：1、长度：18-25； 2、GC含量：40-60％，正反向引物相差不要大于5%； 3、Tm值：55以上（到65），实在不行50以上也可以，正反向引物相差不要大 于5； 4、3’端结尾最好是GC，其次是T，不要A； 5、正反向引物连续配对数小于4； 6、在NCBI上的Primer Blast上看引物特异性如何； （如果克隆的话不能满足条件也没办法。） 不是必须条件，但可以考虑：多个基因设计引物时，可尽量使Tm值相似，方便PCR。 步骤： 一、打开PrimerSelect和EditSeq。 二、在EditSeq中输入你的序列。 引物有一对F和R 1、对于F是从5’到3’，在序列的前部分选择长度为18-25bp的碱基，如果你是要验证就随便选，如果你是要克隆就在最开始选，不符合原则就只能在你选的后边增或减碱基。 2、将选择的F引物输入到PrimerSelect中，在File中选择Enter New Primer，复制，OK，然后可以看到引物的情况，看看长度、Tm、GC含量是不是符合标准，不符合就继续选。 3、对于R是从3’到5’，选中序列，在EditSeq的Goodies中选择第一个“反向互补”，此时序列已反向互补，按照前面F的方法搜索R的引物。、 4、注意你想要的目的带的大小，比如序列是1000bp，你想PCR出来800大小的目的带，那
就要看看F和R之间的长度在你想要的范围内。可以将R反向互补，在正向的序列中搜索R 在的位置，就是在EditSeq中选择Search，点击第一个Find，开始搜寻。 5、搜索完引物在PrimerSelec中的Report中选择前两个查看二聚体情况。 6、在NCBI上的Primer Blast上看引物特异性如何。 7、因为是克隆，所以引物要有酶切位点，酶切位点的加入主要考虑所用到的表达载体，在NEBcutter网站中输入总序列查看可用的酶切位点。在引物上游加入酶切位点，注意加入时载体的表达的方向，前面的酶切位点在引物F上，后面的酶切位点在引物R上。一般在引物上游还要加上两个保护碱基。 2、提取醋栗DNA 3、PCR扩增与目的基因回收 PCR先找合适的退火温度，找到后回收时就可以多PCR几管，一般我们用20ul的体系，PCR5管就可以回收，就是琼脂糖凝胶回收，将目的基因用刀片切下来，用试剂盒回收。回收完可以再跑电泳检测一遍。 PCR： 20ul体系：灭菌水，若模板为质粒灭菌水； ； 10乘Taq ； 引物F、R各； 模板1ul；若为质粒就够； 最后加入Taq酶，Taq酶现用现拿。 过程：我们用的是预变性94℃3min； 然后进入循环（要进行克隆的话循环数最好不要超过30个），循环过程：变性94℃30s，退火退火温度下30s，延伸72℃时间根据目的基因长度和酶而定，一般Taq酶30s可以延伸1kb； 循环完成后，此时尚有正处于合成过程中的dna链,为了保证充分的得率,所以再增加7分钟的72℃延伸； 最后4℃保存待用。
实验：目的基因克隆（PCR技术） 【课前预习】 PCR (polymerase chain reaction) 反应的基本原理。 【目的要求】 1．学习和掌握PCR 反应的基本原理与实验技术方法。 2．认真完成每一步实验操作，详细记录实验现象和结果并加以分析和总结。 【基本原理】 类似于DNA 的天然复制过程，其特异性依赖于与靶序列两端互补的寡核苷酸引物。PCR 由变性--退火--延伸三个基本反应步骤构成：①模板DNA的变性：模板DNA 经加热至93℃左右一定时间后，使模板DNA双链或经PCR 扩增形成的双链DNA 解离，使之成为单链，以便它与引物结合，为下轮反应作准备；②模板DNA 与引物的退火(复性)：模板DNA 经加热变性成单链后，温度降至55℃左右，引物与模板DNA 单链的互补序列配对结合；③引物的延伸：DNA 模板--引物结合物在TaqDNA 聚合酶的作用下，以dNTP为反应原料，靶序列为模板，按碱基配对与半保留复制原理，合成一条新的与模板DNA 链互补的半保留复制链重复循环变性--退火--延伸三过程，就可获得更多的“半保留复制链”，而且这种新链又可成为下次循环的模板。每完成一个循环需2～4 分钟，2～3 小时就能将待扩目的基因扩增放大几百万倍。到达平台期(Plateau)所需循环次数取决于样品中模板的拷贝。【实验用品】 1．材料：重组质粒DNA作为模板 2．器材和仪器：移液器及吸头，硅烷化的PCR 小管，DNA扩增仪(PE 公司)，琼脂糖凝胶电泳所需设备(电泳槽及电泳仪)，台式高速离心机 3．试剂： ①10×PCR 反应缓冲液：500mmol/L KCl, 100mmol/L Tris·Cl, 在25℃下, pH9.0, 1.0％Triton X-100。 ②MgCl2 ：25mmol/L。 ③ 4 种dNTP 混合物:每种 2.5mmol/L。 ④Taq DNA聚合酶5U/μl。 ⑤T4 DNA连接酶及连接缓冲液：
酪氨酸激酶体系：胰岛素，生长激素，促红细胞生成素 记忆：为了生计老暗算别人一刀，简直是个畜生 血沉ESR加快：纤维蛋白质，球蛋白，胆固醇 记忆：单纯（胆固醇）少女求（球蛋白）签（纤维蛋白）问红尘（红细胞血沉） 单核细胞：3-8%,中性粒50-70%,淋巴20-40% 记忆：单身三八，中年无（5）妻（7），聆听儿诗 红细胞生成素调节：BPA(爆式促进激活物)，促红细胞生成素，性激素，生长激素，甲状腺素 记忆：一个畜生（促红细胞生成素）居然对生（生长激素）怀六甲（甲状腺素）的女人实施性（性激素）暴力， 性激素：雄激素是正性，雌激素负性 铅中毒：动力性肠梗阻，卟啉症 记忆：铅可抑制ALA脱水酶和亚铁鳌合酶的活性 蛋白质大部分是由肝脏合成的，除了Y-球蛋白是由浆细胞合成的 记忆：将（浆）在外（Y）,君令有所不受 失读症：角回受损-->独角戏（歌名）
感觉性失语症：颞上回受损-->驱赶余孽 运动性失语症：Broca区-->洞与B联系起来，或Bro像brother,brother爱运动啊 快痛-->传入纤维是A*纤维，慢痛C纤维， 记忆：形状像豆芽，豆芽长的快，C纤维-->chronic是慢的意思 凝血因子：IV->钙离子，V->易变因子 记忆：武艺盖世 凝血辅因子：IV，V，III，VIII 记忆：我师傅三八 被消耗的因子：II，V，VII，VIII 记忆：浩霸占我妻儿 内源性凝血：启动因子12,慢，单独凝血因子12,11,9,8,4， 外源性凝血：启动因子3,快，单独凝血因子，3,4,7 记忆：123,3+4=7
生理性抗凝血酶III：丝氨酸酶抑制物-->9-12因子 记忆：3+9=12 VitA的活性形式：视黄醇，视黄醛，视黄酸 记忆：看（视）黄色的电影是看A片 几乎所有的血浆蛋白均为糖蛋白，除了清蛋白，清蛋白与胆红素结合 记忆：因为清蛋白清高，不愿与糖为伍， 清蛋白-->清扫有毒物质-->与胆红素结合-->肝脏-->胆红素与Y,Z蛋白结合 对凝血酶敏感的凝血因子：I,V,VIII,XIII 蛋白质C系统：蛋白质C(PC),凝血酶调节蛋白，蛋白质S,蛋白C抑制物 蛋白质C (PC):灭活Va,VIIIa，抑制Fx及凝血酶原激活 记忆：我爸（5,8）嫖娼（P,C） 蛋白C抑制物与PC形成APC 肝脏是储存维生素A,E,B12,K的主要场所 记忆：干(肝)脆罢课（BAKE） 氨基酸的记忆： 中性氨基酸：中性氨基酸：谷氨酰胺，天冬酰胺，酪氨酸，丝氨酸，色氨酸，苏氨酸，胱氨酸，蛋氨酸 记忆：(中)国(股东老实)好(色输)成穷(光蛋)
论著 文章编号:1007-8738(2004)02-0230-04 非受体型酪氨酸激酶-Syk 蛋白的提取与纯化 单保恩1,董 青1,李宏芬1,陈 晶1,董金琢2,马 洪2 (1河北医科大学第四医院科研中心,河北石家庄050011;2美国MD 技术公司,Mo 63021,美国) 收稿日期:2003-06-07; 修回日期:2003-09-18基金项目:美国MD 技术公司合作课题基金资助(2002年)作者简介:单保恩(1962-),男,河北邯郸人,教授,博士生导师. Tel:(0311)6033941-290;Email:baoenshan@yahoo.c https://www.doczj.com/doc/af5507622.html, Extraction and purification of non -recep -tor type PTKs -Syk SHAN Bao -en 1,DO NG Qing 1,LI Hong -fen 1 ,C HE N Jing 1,DO NG Jin -zhuo 2,MA Hong 2 1 Research Center ,The Fourth Hospital of Hebei Medical University,Shijiazhuang 050011,China;2MD Technologies Inc.845Pheasant Woods Drive Manchester,Mo 63021,USA Abstract AIM:To extract and purify Syk protein from Sf 21cells transfec-t ed by syk gene.METHODS:Sf 21cells were transfected with recom bi nant syk gene.After 48h of incubation at 28 ,the transfected cells were collected and sonicated with Sonfier son-i cator on ice.Filtered cell extract was loaded onto a R eactive Yellow -3res i n column and Toyopearl AF -H eparin -650M colum n respectively.The character of Syk protein in the fractions were i dentified by SDS -PAGE,W estern blotting and IEF.RESULTS:225m g of protein containing Syk were obtained from Sf 21cells (2.5 109)extract.There were two subpopulations in the elu -tion of Reactive Yellow -3resin column with the same relative molecular mass (M r )72 103.The two subpopulations were then applied on Toyopearl AF -H eparin -650M column and two pure proteins were obtained.The results of SDS -PAGE,W es-t ern blotting,and IEF showed the two proteins having the sam e relative molecular mass (72 103),corresponding to Syk,but with different pI.CONCLUSION:The y ield of Syk was 8m g from 2.5billion cells and the purity was >95%.The two purified Syk proteins have the sam e M r and different pI.The purified Syk protein can be applied to study Syk s m echanism,produce ant -i Syk antibody and invent Syk diagnosis kit,etc .Keywords:Syk;purification;chromatography;Sf 21cells 摘要 目的:从转染syk 基因的Sf21细胞中提取、纯化免疫相关因子 Syk 蛋白。方法:将syk 基因转染Sf21细胞,于28 培养48h,收集细胞,用超声波破碎仪裂解细胞,提取裂解液中总蛋白,用Yellow -3凝胶和Toyopear-l AF -Heptin -650M 凝胶层析柱分离、纯化。层析液中的Syk 蛋白存在和性质,用SDS -PAGE 、免疫印迹实验和等电聚焦实验鉴定。结果:从25亿个Sf21细胞裂解液中提取了含有Syk 的225mg 蛋白质。经Yellow -3凝胶层析分离,得到两个亚种的Syk 蛋白,相对分子质量(M r )均为72 103。进一步用Toyopear-l AF -Heptin -650M 凝胶层析纯化后,得到两个纯的Syk 蛋白,SDS -PAGE 、免疫印迹实验结果显示,两种Syk 的M r 均为72 103,与Syk 的理论相对分子质量吻合。但等电聚焦实验显示,这两种Syk 蛋白成分具有不同的pI 值。结论:从25亿个转染syk 基因的Sf21细胞中纯化出8mg Syk 蛋白,纯度高于95%。这两种Syk 的M r 虽然相同,但具有不同的pI 值,是两个亚种。这些Syk 可用于研究Syk 的作用机制、抗Syk 抗体的制备和Syk 诊断试剂盒的制备等。关键词:Syk;分离纯化;凝胶层析;Sf21细胞中图分类号:R392.11 文献标识码:B 非受体型酪氨酸激酶(spleen tyrosine kinase,Syk),是一种B 细胞激活信号转导中最重要的激酶 [1] ,与T 细胞激活中的ZAP -70属于同一个PTK 家 族,M r 为72 103。Syk 在T 细胞和B 细胞的成熟和活化过程中起关键作用[2]。该酶除有激酶活性中心SH1之外,还有两个SH2结构域,因而成为磷酸化I -TAM 招募的首选对象。被招募的Syk 立即成为Src 作用的第二个靶目标,进而启动B 细胞活化信号转导的三条主要途径(磷脂酰肌醇途径、MAP 激酶相关途径和磷酸肌醇3激酶途径),激活各种转录因子转位进入细胞核,与基因启动子区域中各种顺式作用元件或DNA 小盒结合,使相应基因发生转录激活和产物表达,调整B 细胞等细胞克隆的蛋白质表达、细胞分化或凋亡。研究发现,Syk 不但是免疫调节因子,在肿瘤发生发展中也发挥着重要作用。但是,用于研究Syk 的蛋白标准品、抗体和诊断用试剂很难取得。我们介绍从转染s yk 基因的Sf21细胞中提取、纯化免疫相关因子Syk 蛋白。