CN102276729A - Antibacterial peptide bovine lactoferricin-thanatin (LF-TH) and Escherichia coli recombination preparation method thereof - Google Patents
Antibacterial peptide bovine lactoferricin-thanatin (LF-TH) and Escherichia coli recombination preparation method thereof Download PDFInfo
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Abstract
The invention relates to an antibacterial peptide bovine lactoferricin-thanatin (LF-TH) and an Escherichia coli recombination preparation method thereof. By the antibacterial peptide LF-TH, the problem of low antibacterial activity of the conventional antibacterial peptide is solved. An amino acid sequence of the antibacterial peptide LF-TH comprises 33 amino acids and is shown by SEQ ID No. 1, and a sequence of a coding gene of the antibacterial peptide is shown by SEQ ID No. 2. The method comprises the following steps of: 1, designing the antibacterial peptide LF-TH and the coding gene of the antibacterial peptide LF-TH; and 2, connecting the coding gene of the antibacterial peptide LF-TH and plasmid to construct a recombinant Escherichia coli expression vector, transforming E. coliBL21(DE3), performing induced expression and purifying to obtain fusion protein, cracking the fusion protein by enterokinase to release the antibacterial peptide LF-TH, and purifying to obtain the antibacterial peptide LF-TH. The antibacterial peptide LF-TH has high antibacterial activity, and has an obvious inhibiting effect on staphylococcus aureus, Escherichia coli, pseudomonas aeruginosa and salmonella typhimurium.
Description
Technical field
The present invention relates to antibacterial peptide and preparation method thereof.
Background technology
Microbiotic is extensive use of a series of problems such as flora imbalance, the increase of Resistant strain and the counter productives such as toxic side effects of medicine brought for a long time and is on the rise, especially resistant strains has become the insurmountable problem of microbiotic, and research and development novel and effective antibacterials have become the task of top priority.Antibacterial peptide is the polypeptide that a class has important immunization in the organism, is the important component part of body non-specific immune function.Antibacterial peptide has characteristics such as molecular weight is little, thermally-stabilised, good water solubility, has a broad antifungal spectrum.Antibacterial peptide has unique antibiotic mechanism of action that is different from, and bacterial strain is difficult for it is produced resistance.Antibacterial peptide is because self good characteristic becomes a kind of research and development focus of new antibacterials.
The price of antibacterial peptide is the restricted bottleneck that the puzzlement antibacterial peptide is used with preparation, utilizes gene engineering method reorganization preparation antibacterial peptide to provide effective means for solving the antibacterial peptide scale preparation.Up to the present, have been found that above more than 1000 kind of antibacterial peptide at occurring in nature.Yet the natural antibacterial peptide anti-microbial activity is not strong, and some antibacterial peptide has cytotoxicity to eukaryotic cells, causes the host animal erythrocyte hemolysis.For this reason, the researchist improves the biological function of antibacterial peptide molecule by many diverse ways and means, as different antibacterial peptide molecules are carried out heterozygosis, with the brachymemma of antibacterial peptide molecule, amino acid replace, C end amidation etc. all is method commonly used in improving antibacterial peptide molecular biology activity research.Many studies show that, the heterozygosis of different antibacterial peptide molecules are the effective ways that improve antibacterial peptide activity, elimination or reduce antibacterial peptide cytotoxicity and hemolytic activity, often are used in research antibacterial peptide molecule structure activity relationship and molecular designing.
(Bovine Lactoferricin is the cationic antibacterial peptide that the Bovinelactoferrin degraded obtains LfcinB) to lactoferrin, extensively is present in multiple tissue of Mammals and mucous layer, participates in the non-specific humoral immunization of body.Obtain separation in 1992 by Bellamy the earliest, its anti-microbial activity is stronger more than 400 times than Bovinelactoferrin, have that immunogenicity is little, good water solubility, broad-spectrum sterilization in addition can fungicidal, advantage such as protozoon, can tolerate the degraded of proteolytic enzyme and peptase in the gi tract.It is dead plain that (Thanatin is one of the widest antibacterial peptide of the antimicrobial spectrum found in insect spot ventral spine benefit stinkbug Th), and Gram-positive, Gram-negative bacteria and fungi are all had effect, but little to the yeast influence.Dead element does not have hemolytic to mammalian cell.
Summary of the invention
The objective of the invention is to have the not high problem of antibacterial peptide anti-microbial activity now, and antibacterial peptide LF-TH and intestinal bacteria recombination and preparation thereof are provided in order to solve.
The aminoacid sequence of antibacterial peptide LF-TH comprises 33 amino acid, its aminoacid sequence is as follows: PheLysCysArgArgTrpGlnTrpArgTrpLysLysLeuGlyAlaLysProValPr oIleIleTyrCysAsnArgArgThrGlyLysCysGlnArgMet (being FKCRRWQWRWKKLGAKPVPIIYCNRRTGKCQRM), its coding gene sequence is shown in SEQ ID No.2.
The intestinal bacteria recombination and preparation of antibacterial peptide LF-TH carries out according to the following steps:
One, chooses the 1st～15 amino acids sequence Lfcin B (1～15) of LfcinB, the tenth amino acids wherein replaces with Trp by Met, choose the 4th～21 amino acids sequence Th (4～21) of Thanatin, Lfcin B (1～15) is connected with Th (4～21), design antibacterial peptide LF-TH, design the encoding gene of antibacterial peptide LF-TH then according to the e. coli codon preferences;
Two, the encoding gene with antibacterial peptide LF-TH is connected with plasmid pET-32a (+), make up the expression of recombinant e. coli carrier, transformed host cell E.coli BL21 (DE3), behind the IPTG abduction delivering, centrifugal collection thalline, ultrasonic treatment, centrifugal collection supernatant liquor, utilize Ni-NTA chromatography column, protein purification instrument to obtain the purpose recombination fusion protein, enteropeptidase cracking purpose recombination fusion protein discharges antibacterial peptide LF-TH then, behind the RP-HPLC purifying, obtain antibacterial peptide LF-TH, promptly finish the intestinal bacteria reorganization preparation of antibacterial peptide LF-TH;
Wherein the aminoacid sequence of antibacterial peptide LF-TH comprises 33 amino acid in the step 1, and its aminoacid sequence is as follows: PheLysCysArgArgTrpGlnTrpArgTrpLysLysLeuGlyAlaLysProValPr oIleIleTyrCysAsnArgArgThrGlyLysCysGlnArgMet;
The coding gene sequence of antibacterial peptide LF-TH is shown in SEQ ID No.2 in the step 1.
The antibacterial peptide LF-TH anti-microbial activity height of the present invention's preparation, streptococcus aureus, intestinal bacteria, Pseudomonas aeruginosa and Salmonella typhimurium had the obvious suppression effect, anti-microbial activity obviously improves, and has widened antimicrobial spectrum, and acellular hemolytic and animal toxicity.The present invention utilizes escherichia expression system successfully to realize the reorganization preparation, having obtained anti-microbial activity and antimicrobial spectrum all has the antibacterial peptide LF-TH of improvement, the Biological resources of antibacterial peptide have been widened, has wide application potential aspect the development of medicine, veterinary drug, fodder additives, food preservative and the exploitation, and be active better high-efficiency antimicrobial peptide molecule of screening and genetically engineered preparation thereof, realize finally that the industrialization of antibacterial peptide is cheap to produce that to seek the theory and technology route significant.
Description of drawings
Fig. 1 is the gel electrophoresis figure of the encoding gene of antibacterial peptide LF-TH in the embodiment two, and wherein swimming lane 1 is represented dna molecular amount standard, the encoding gene of swimming lane 2 expression antibacterial peptide LF-TH; Fig. 2 screens the gel electrophoresis figure of positive recombinant plasmid for PCR method in the embodiment two, wherein swimming lane 1 is represented dna molecular amount standard, swimming lane 2 expressions contain the pcr amplification product of the bacterium colony of recombinant plasmid, and swimming lane 3 expressions contain the pcr amplification product of the bacterium colony of pET-32a (+); Fig. 3 is the gel electrophoresis figure of IPTG abduction delivering in the embodiment two, wherein swimming lane 1 is represented protein molecular weight standard, swimming lane 2 is represented not inductive recombinant expression plasmid Transformed E .coli BL21 (DE3) expression product, swimming lane 3 expression pET-32a (+) Transformed E .coli BL21 (DE3) abduction delivering products, swimming lane 4 expression recombinant expression plasmid Transformed E .coliBL21 (DE3) abduction delivering products, the lysing cell supernatant liquor of swimming lane 5 expression recombinant expression plasmid Transformed E .coli BL21 (DE3) abduction deliverings; Fig. 4 is the gel electrophoresis figure of purpose recombination fusion protein in the embodiment two, and wherein swimming lane 1 is represented protein molecular weight standard, the fusion rotein of swimming lane 2 expression purifying; Fig. 5 is the gel electrophoresis figure of enteropeptidase cracking purpose recombination fusion protein in the embodiment two, and wherein swimming lane 1 is represented enteropeptidase cracked fusion rotein, swimming lane 2 expression protein molecular weight standards; Fig. 6 is the gel electrophoresis figure that obtains antibacterial peptide LF-TH in the embodiment two behind the purifying, and wherein swimming lane 1 is represented protein molecular weight standard, swimming lane 2 expression LF-TH; Fig. 7 is the analysis chart that electron spray mass spectrometry is measured the molecular weight of peptide in the embodiment two.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the aminoacid sequence of present embodiment antibacterial peptide LF-TH comprises 33 amino acid, its aminoacid sequence is as follows: PheLysCysArgArgTrpGlnTrpArgTrpLysLysLeuGlyAlaLysProValPr oIleIleTyrCysAsnArgArgThrGlyLysCysGlnArgMet, its coding gene sequence is as follows: TTCAAATGCCGTCGTTGGCAGTGGCGTTGGAAAAAACTGGGTGCGAAACCGGTTCC GATCATCTACTGCAACCGTCGTACCGGTAAATGCCAGCGTATG.
Embodiment two: the intestinal bacteria recombination and preparation of present embodiment antibacterial peptide LF-TH carries out according to the following steps:
One, chooses the 1st～15 amino acids sequence Lfcin B (1～15) of LfcinB, the tenth amino acids wherein replaces with Trp by Met, choose the 4th～21 amino acids sequence Th (4～21) of Thanatin, Lfcin B (1～15) is connected with Th (4～21), design antibacterial peptide LF-TH, design the encoding gene of antibacterial peptide LF-TH then according to the e. coli codon preferences;
Two, the encoding gene with antibacterial peptide LF-TH is connected with plasmid pET-32a (+), make up the expression of recombinant e. coli carrier, transformed host cell E.coli BL21 (DE3), behind the IPTG abduction delivering, centrifugal collection thalline, ultrasonic treatment, centrifugal collection supernatant liquor, utilize Ni-NTA chromatography column, protein purification instrument to obtain the purpose recombination fusion protein, enteropeptidase cracking purpose recombination fusion protein discharges antibacterial peptide LF-TH then, behind the RP-HPLC purifying, obtain antibacterial peptide LF-TH, promptly finish the intestinal bacteria reorganization preparation of antibacterial peptide LF-TH;
Wherein the aminoacid sequence of antibacterial peptide LF-TH comprises 33 amino acid in the step 1, and its aminoacid sequence is as follows: PheLysCysArgArgTrp GlnTrpArgTrpLysLysLeuGlyAlaLysProValProIleIleTyrCysAsnAr gArgThrGlyLysCysGlnArgMet;
The coding gene sequence of antibacterial peptide LF-TH is shown in SEQ ID No.2 in the step 1.
According to the encoding gene of e. coli codon preferences design antibacterial peptide LF-TH, process is as follows: the sticky end that adds restriction enzyme Nco I, EcoR I respectively in the upstream and the downstream of gene in the present embodiment step 1; Five codons that add the recognition site (AspAspAspAspLys, i.e. DDDDK) of coding enteropeptidase before the encoding gene fragment, normal chain are 5 '-
GACGATGACGACAAATTCAAATGCCGTCGTTGGCAGTGGCGTTGGAAAAAACTGGGTGCGAAACCGGTTCC GATCATCTACTGCAACCGTCGTACCGGTAAATGCCAGCGTATG
G-3 ', minus strand is 5 '-
CATACGCTGGCATTTACCGGTACGACGGTTGCAGTAGATGATCGGAACCGGTTTCG CACCCAGTTTTTTCCAACGCCACTGCCAACGACGGCATTTGAA
TTT GTCGT CATCGTC-3 ';
Italicized item is restriction restriction endonuclease Nco I, EcoR I sticky end sequence, and underscore partly is the codon of DDDDK, two terminator codons for adding in the square frame; Equimolar positive anti-chain is mixed, annealing, 2% agarose gel electrophoresis after purification kit reclaims, obtains the gene fragment of purifying; The entire segment design length is 130bp, as shown in Figure 1.
Make up the expression of recombinant e. coli carrier in the present embodiment step 2, process is as follows: the encoding gene of antibacterial peptide LF-TH is connected between the Nco I, EcoR I site of plasmid pET-32a (+), connect product transformed host cell E.coliDH5 α, the PCR method screening positive clone, forward primer P1:5 '-CCCAAGGGGTTATGCTAGTT-3 ', reverse primer P2:5 '-TGCACCATCATCATCATCAT-3 ', reaction conditions: 94 ℃ of 5min; 95 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 1min, 32 circulations are extended 5min for back 72 ℃; The primer P1 and P2 respectively with 56-75, the complementation of 308-327 bit base of pET-32a (+), 2% agarose gel electrophoresis detects, The selection result as shown in Figure 2, positive control (bacterium colony that contains maternal plasmid pET-32a (+)) PCR product is the DNA band of 270bp, and the amplified production of positive recombinant plasmid is the DNA band about 380bp, further the dna sequencing result shows, positive recombinant vectors base sequence is in full accord with design, has successfully made up recombinant expression plasmid pELT.
IPTG abduction delivering in the present embodiment step 2, process is as follows: with expression of recombinant e. coli carrier Transformed E .coliBL21 (DE3), positive transformant is inoculated in the 5ml LB substratum and (contains 100 μ g/ml penbritins), 37 ℃ of shaking table overnight incubation, transfer in the 50ml same medium with 1% amount, 37 ℃ are cultured to OD
600=0.6, add IPTG to final concentration 0.3mmol/L, induce 4h for 30 ℃, sampling 15%SDS-PAGE analyzes; As shown in Figure 3, be about 21kDa, conform to, and the fusion rotein of expressing major part among E.coliBL21 (DE3) exists with soluble form with the expection size through the recombination fusion protein relative molecular mass of IPTG abduction delivering.
Centrifugal collection thalline in the present embodiment step 2, ultrasonic treatment, centrifugal collection supernatant liquor, utilize Ni-NTA chromatography column, protein purification instrument to obtain the purpose recombination fusion protein, process is as follows: gained inducing culture thing is through the centrifugal 10min of 4000r/min behind the IPTG abduction delivering, collect thalline, PBS (140mM NaCl, 2.7mM KCl, 10mM Na
2HPO
4, 1.8mM KH
2PO
4PH 7.3) washing is once, the resuspended thalline of amount that adds 5ml PBS according to the 100ml culture, ultrasonication, 4 ℃ of centrifugal 30min of 12000r/min collect supernatant, Ni-NTA chromatography column purifying target protein, the SDS-PAGE analysis is carried out in sampling, the gel thin-layer scanning analysis, and the purity of fusion rotein is greater than 90% (as Fig. 4); Protein purification instrument (GE, the U.S.) carries out desalination to eluted protein, collects main protein peak, utilizes the Bradford method to measure protein content, calculates the productive rate of fusion rotein, and average every liter of substratum can obtain about 40mg fusion rotein; Obtain albumen dry powder after the vacuum lyophilization.
Enteropeptidase cracking purpose recombination fusion protein discharges antibacterial peptide LF-TH in the present embodiment step 2, behind the RP-HPLC purifying, obtain antibacterial peptide LF-TH, process is as follows: enteropeptidase damping fluid (150mM Tris-HCl, 150mM NaCl, 2.5mM CaCl2, pH 7.4) albumen is soluble and fused, adjustment concentration is 1mg/ml, every milligram of fusion rotein adds the 8U enteropeptidase, 21 ℃ of effect 16h, sampling Tricine-SDS-PAGE analyzes, and the result is as shown in Figure 5, be about 17 and the 4kD place have protein band, successfully discharged LF-TH.Lysate is crossed the Ni-NTA chromatography column and is removed label protein and uncracked fusion rotein, collects effluent liquid.RP-HPLC purifying LF-TH, chromatographic condition is: Platisil C18 chromatography column (250cm, 4.6mm, 5 μ m,
), column temperature: 25 ℃; Mobile phase A is a 0.1%TFA/ water; Mobile phase B is the 0.1%TFA/ acetonitrile, and elution program is: 0～25～30min, 20%～45%～100%A; Flow velocity 1mL/min; Detect wavelength: 214nm.Collect elution peak, vacuum freezedrying, through Tricine-SDS-PAGE, the result has obvious band at the 4kD place as shown in Figure 6.The Bradford method is measured concentration, and average every liter of substratum can obtain about 500 μ g antibacterial peptides.Use electron spray mass spectrometry and measure the molecular weight of peptide, as shown in Figure 7, determining molecular weight is 4195.14, and is consistent with the LF-TH theoretical molecular.
Present embodiment prepares gained antibacterial peptide LF-TH, determination of activity:
With intestinal bacteria (ATCC25922), streptococcus aureus (ATCC25923), Salmonellas (CVCC533), Salmonella typhimurium (C77-31) is indicator, the minimal inhibitory concentration of micro-broth dilution method LF-TH (Mininum Inhibitory Concentration, MIC), measure the MIC of LfcinB 15, Thanatin simultaneously.(the test bacterium is inoculated in incubated overnight in the MHA substratum to damping fluid for 0.02% acetate, 0.4%BSA) dilution antibacterial peptide.The overnight culture that the MHB substratum will be tested bacterium is diluted to 2 * 10
6CFU/ml.Bacterium liquid is added to 96 porocyte culture plates, the hole, 1 hole to 11 of every row, every hole 100 μ l, No. 12 the hole adds the fresh MHB substratum of 100 μ l as blank.The antibacterial peptide stock solution of different concns is added in the 1-10 hole.No. 11 the hole does not add antibacterial peptide, as positive control.37 ℃ of shaking table 120rpm constant temperature culture 18 hours, microplate reader is surveyed the 490nm light absorption value.Absorbancy is defined as the MIC of this peptide to this test bacterium than the concentration of the antibacterial peptide in the low hole more than 50%, No. 11 holes.By table 1 as seen, present embodiment prepares four kinds of tests of gained antibacterial peptide LF-TH bacterium and all has the obvious suppression effect, and intestinal bacteria, Salmonellas, Salmonella typhimurium (C77-31) anti-microbial activity are obviously improved than LfcinB.Compare with Thanatin, the activity of streptococcus aureus, Pseudomonas aeruginosa is significantly improved.
Table 1
Present embodiment prepares gained antibacterial peptide LF-TH, and hemolytic is measured:
Gather normal human blood 1mL, be dissolved into behind the anticoagulant heparin in the 2ml PBS solution, the centrifugal 5min of 1000g collects red corpuscle; With PBS washing 3 times, use 10ml PBS resuspended again, get 100 μ L red cell suspensions and 100 μ L and mix with the antibacterial peptide solution of the above-mentioned different concns of PBS dissolved, constant temperature is hatched 1h in 37 ℃ of incubators; Take out behind the 1h, 4 ℃, the centrifugal 5min of 1000g take out supernatant liquor and use microplate reader in 540nm place photometry absorption value, average for every group, and comparative analysis.100 μ l PBS are as negative control; 100 μ l 0.2%Tritonx-100 do positive control.Detected result is that present embodiment prepares gained antibacterial peptide LF-TH does not have hemolytic activity under 256 μ g/ml concentration.
Present embodiment prepares gained antibacterial peptide LF-TH, acute toxicity test in the mouse body:
Adopt 40 of Kunming white mouse, male and female half and half, body weight 25 ± 0.31 according to 1mg/kg dosage intramuscular injection antibacterial peptide LF-TH, once a day, continuous 7 days, is observed the mouse toxicity reaction.The acute toxicity tests shows in the animal body of antibacterial peptide LF-TH, and intramuscular injection antibacterial peptide LF-TH is after 7 days, the no abnormal reaction of mouse, and movable normal, 40 mouse all survive, and prove that present embodiment prepares gained antibacterial peptide LF-TH and has no side effect.
More than used main agents and material: e. coli bl21 (DE3), DH5 α, streptococcus aureus ATCC25923, intestinal bacteria ATCC25922, Salmonella typhimurium C77-31, Pseudomonas aeruginosa (ATCC27853), plasmid pET-32a (+) are that Animal nutrition institute of Northeast Agricultural University preserves.T4DNA ligase enzyme, restriction enzyme NcoI, EcoR I, dNTP, Taq archaeal dna polymerase are available from precious biotechnology (Dalian) company limited, Tryptones, yeast extract are purchased the company in Oxoid, DNA Marker, low molecular weight of albumen standard are all available from TIANGEN Biotech (Beijing) Co., Ltd., the Ni-NTA chromatography column is purchased the company in Novegen, and other reagent are homemade analytical pure.Polynucleotide passage is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.LfcinB, Thanatin (purity＞98%) are synthetic by company of the biochemical company limited of Shanghai gill.In concrete operations, relate to the equal by specification operation of various reaction reagents of use.
Claims (2)
1. antibacterial peptide LF-TH, the aminoacid sequence that it is characterized in that antibacterial peptide LF-TH comprises 33 amino acid, its aminoacid sequence is as follows: PheLysCysArgArgTrpGlnTrpArgTrpLysLysLeuGlyAlaLysProValPr oIleIleTyrCysAsnArgArgThrGlyLysCysGlnArgMet, its coding gene sequence is shown in SEQ IDNo.2.
2. the intestinal bacteria reorganization prepares the method for antibacterial peptide LF-TH according to claim 1, it is characterized in that the intestinal bacteria recombination and preparation of antibacterial peptide LF-TH carries out according to the following steps:
One, chooses the 1st～15 amino acids sequence Lfcin B (1～15) of LfcinB, the tenth amino acids wherein replaces with Trp by Met, choose the 4th～21 amino acids sequence Th (4～21) of Thanatin, Lfcin B (1～15) is connected with Th (4～21), design antibacterial peptide LF-TH, design the encoding gene of antibacterial peptide LF-TH then according to the e. coli codon preferences;
Two, the encoding gene with antibacterial peptide LF-TH is connected with plasmid pET-32a (+), make up the expression of recombinant e. coli carrier, transformed host cell E.coli BL21 (DE3), behind the IPTG abduction delivering, centrifugal collection thalline, ultrasonic treatment, centrifugal collection supernatant liquor, utilize Ni-NTA chromatography column, protein purification instrument to obtain the purpose recombination fusion protein, enteropeptidase cracking purpose recombination fusion protein discharges antibacterial peptide LF-TH then, behind the RP-HPLC purifying, obtain antibacterial peptide LF-TH, promptly finish the intestinal bacteria reorganization preparation of antibacterial peptide LF-TH;
Wherein the aminoacid sequence of antibacterial peptide LF-TH comprises 33 amino acid in the step 1, and its aminoacid sequence is as follows: PheLysCysArgArgTrpGlnTrpArgTrpLysLysLeuGlyAlaLysProValPr oIleIleTyrCysAsnArgArgThrGlyLysCysGlnArgMet;
The coding gene sequence of antibacterial peptide LF-TH is shown in SEQ ID No.2 in the step 1.
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| CN102863539A (en) * | 2012-10-11 | 2013-01-09 | 东北农业大学 | Fusion tandem antibacterial peptide and preparation method thereof |
| CN103145848A (en) * | 2013-02-06 | 2013-06-12 | 上海高龙生物科技有限公司 | Recombinant lactoferrin dipeptide, expression vector and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102816206A (en) * | 2012-08-27 | 2012-12-12 | 重庆市畜牧科学院 | Synthetic peptide and application thereof |
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| CN102863539B (en) * | 2012-10-11 | 2014-12-17 | 东北农业大学 | Fusion tandem antibacterial peptide and preparation method thereof |
| CN103145848A (en) * | 2013-02-06 | 2013-06-12 | 上海高龙生物科技有限公司 | Recombinant lactoferrin dipeptide, expression vector and application thereof |
| CN103145848B (en) * | 2013-02-06 | 2014-11-26 | 上海高龙生物科技有限公司 | Recombinant lactoferrin dipeptide, expression vector and application thereof |
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