CN104789625A - Method for secretory expression of human alpha defensin 5 in saccharomyces cerevisiae - Google Patents
Method for secretory expression of human alpha defensin 5 in saccharomyces cerevisiae Download PDFInfo
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- CN104789625A CN104789625A CN201510230559.8A CN201510230559A CN104789625A CN 104789625 A CN104789625 A CN 104789625A CN 201510230559 A CN201510230559 A CN 201510230559A CN 104789625 A CN104789625 A CN 104789625A
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Abstract
The invention discloses a method for secretory expression of human alpha defensin 5 in saccharomyces cerevisiae. The method comprises the following steps: constructing a recombinant saccharomyces cerevisiae expression vector containing human alpha defensin 5 gene by using a secreting type saccharomyces cerevisiae expression vector, introducing the constructed recombinant saccharomyces cerevisiae expression vector into the saccharomyces cerevisiae to obtain the recombinant saccharomyces cerevisiae, and culturing the recombinant saccharomyces cerevisiae to express the human alpha defensin 5 gene. By adopting the method, the natural saccharomyces cerevisiae with plasmid is taken as an expression system, the target protein polypeptide is directly existed in a supernate of the fermentation solution through the secretory expression, so that the expressed target protein has a correct native conformation; because the product is existed in the supernate, the separation and purification process is greatly simplified without damaging the cell and the production cost is reduced.
Description
Technical field
The present invention relates to a kind of expression method of human α-defensin 5, refer to a kind of method expressing human α-defensin 5 in S. cervisiae expression system especially.
Background technology
Alexin is the class antimicrobial polypeptide active substance produced in organism, exogenous pathogenic agent can be resisted, being subfamily maximum in antibacterial peptide family, is the important component part of organism congenital immunity, constitutes the radical primary immune system of defense in place with complement, Interferon, rabbit etc.
Be similar to general sanitas, natural protection element causes membrane perforation by physical action and reaches the antibacterial effect of broad-spectrum high efficacy, and safe noresidue.Compared with common microbiotic, alexin has certain superiority: one, alexin molecular origin is in living body biological, can enter in microorganism cells well, cause membrane permeability increase and then destroy its energy metabolism system, final activity, the Growth and reproduction realizing suppression microorganism fast.Two, the antimicrobial mechanism of alexin uniqueness makes pathogenic micro-organism not easily produce resistance mutation, safety and reliability.Three, alexin shows and typically acts synergistically with microbiotic, such as: regulate immune response and inflammatory reaction, in and intracellular toxin, regulate tissue injury reparation etc.
In recent years, the effect that the human α-defensin 5 (HD-5) that paneth's cell (Paneth cell, PC) was secreted resists microorganism in intestinal mucosa is subject to extensive concern; HD-5 because of has a broad antifungal spectrum, do not affect intestinal microecology balance, not easily produce the advantage such as resistance, for the anti-enterogenic infection medicine of development of new has welcome dawn., in the 8th article of chromosomal p21-pter district, there are two exons the position of DEFA5, and two exons separate by an intron.DEFA5 gene size is 449bp, wherein front 40bp is that 5' holds non-translational region (5'-UTR), 41 ~ 97bp is the signal peptide sequence of gene, 227 ~ 322bp is only the base sequence of coding HD-5 mature peptide, and the sequence between signal peptide and mature peptide is front fragment, 433 ~ 438 is PolyA sequence.HD-5 precursor protein has 94 amino acid, comprises signal peptide sequence and front region sequence and α-alexin 5 mature peptide.People HD-5 mature peptide (mature human alpha-defensin-5, mHD-5) comprises 32 amino acid, and relative molecular mass is the positively charged ion small peptide of 3.58kD; 6 cysteine residues with conservative property constitute 3 intramolecular disulfide bonds (Cys65-Cys93, Cys72-Cys92, Cys67-Cys82) respectively.
Through further Activity determination, people HD-5 not only can efficiently kill various bacterium, and has stronger antiviral biological activity.Data analysis shows that people HD-5 is likely that in human α-defensin, bacteriostatic activity is the strongest, has significantly press down/kill effect to Gram-negative (G-) bacterium, Gram-positive (G+) bacterium, spirochete, fungi, protozoon and envelope virus etc.People HD-5 also shows stimulates intestinal secretion, suppress NK cytoactive, promote the functions such as inflammatory cell chemotactic, and at anti-human papilloma virus (anti-HPV) (humanpapillomavirus, HPV) infect in the research of human cervical carcinoma cell (HeLa) and there is good biological activity, because this person HD-5 is considered to candidate's newtype drug molecule of viral prevention and treatment of venereal diseases and intestines source property and reproductive tract bacteriological infection, there is good development prospect clinically.But human α-defensin 5 is generally by extracting from particular organization or being obtained by the method for chemically synthesized polypeptide.Therefore, how high-efficiency earth's surface reaches the direction that human α-defensin 5 becomes effort.
Yeast is rapid as engineered receptor bacterium development in recent years, and for intestinal bacteria, yeast has more perfect gene expression regulation mechanism and post translational processing Modifying Capability and secretion capacity, and does not produce intracellular toxin, is good eukaryotic gene recipient bacterium.It is relatively simple that yeast has genetic manipulation, and foreign protein processing is with modification is correct, expression amount is high, be easy to purifying, be suitable for the advantages such as bulk fermentation cultivation.And carry out product specificities design according to prokaryotic organism albumen, target DNA and eukaryotic gene regulatory gene are without homology, therefore there is not the non-specific influences of gene, can the expression of strict regulatory gene, and the expression of regulatory gene is also part less than other system with efficient inducible gene expression artificially.Pichia pastoris phaff is one of Host Strains of conventional expression of recombinant proteins.Be shuttle plasmid for transforming the recombinant plasmid of pichia spp, containing colibacillary replication origin, but this kind of plasmid cannot copy and stable existence in pichia spp, only has that be integrated into by recombinant expression vector could stable existence after in pichia spp gene.Therefore, the factor such as kind and state for the proceeding to of foreign gene, endogenous protease activities, Host Strains can affect the expression efficiency of foreign protein in pichia spp.
Summary of the invention
Technical problem to be solved by this invention is: for above-mentioned the deficiencies in the prior art, provide a kind of can the method for human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae of high expression human α-defensin 5 gene.
In order to solve the problems of the technologies described above, the technical solution adopted in the present invention is: the method for a kind of human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae, the method utilizes secretor type saccharomyces cerevisiae expression to build the recombinant Saccharomyces cerevisiae expression vector containing human α-defensin 5 gene, the recombinant Saccharomyces cerevisiae expression vector built is imported in yeast saccharomyces cerevisiae, obtain recombinant Saccharomyces cerevisiae, cultivate recombinant Saccharomyces cerevisiae and human α-defensin 5 gene is expressed.
Described secretor type saccharomyces cerevisiae expression is pVT102U-α plasmid.The recombinant Saccharomyces cerevisiae expression vector built is pVT102-α-HD5.
The method imported in yeast saccharomyces cerevisiae by the recombinant Saccharomyces cerevisiae expression vector of structure in the present invention is electroporated method, certainly also can adopt other common method in biological field.
The present invention uses the natural yeast saccharomyces cerevisiae that there is plasmid to carry out related experiment as expression system.Relative to pichia spp, yeast saccharomyces cerevisiae has the molecule similar to it and genetic manipulation advantage, but foreign gene unconformability enters host DNA, and do not produce impact in essence to host cell, cell self does not have an impact to foreign protein expression efficiency.And the present invention is secreting, expressing, target protein polypeptide is directly present in the supernatant of fermented liquid, and on the one hand, this phraseology can ensure that the target protein of expressing has correct native conformation; On the other hand, product is present in supernatant, need not smudge cells, enormously simplify the operation of separation and purification, reduces production cost.Moreover yeast saccharomyces cerevisiae is safe bacterial strain, its expression product is easy to the safety requirements reached needed for types of applications, can be widely used in the fields such as food, medicine, Animal nutrition and beauty and health care.
Accompanying drawing explanation
Fig. 1 is the amplification figure that agarose gel electrophoresis detects bridging PCR.
Wherein, 1:20bp DNA Ladder Marker; 2,3: bridging pcr amplification final product.
Fig. 2 is secretor type saccharomyces cerevisiae expression pVT102U-α plasmid map.
Fig. 3 is the electrophoresis detection figure that bacterium colony PCR screens recombinant plasmid pVT102U-α-HD5.
Wherein, 1:Trans2K plus DNA Marker; 2,3: recombinant plasmid positive colony bacterium colony PCR primer.
Fig. 4 is the fungistatic effect figure of HD-5 to streptococcus aureus.
Wherein, the penbritin (Amp) of 1 and 2 expression 100 μ L 0.02mg/mL; The fermented liquid supernatant liquid of 3 and 4 expression 100 μ L recombination yeast S78/pVT102U-α-HD5.
Fig. 5 is that HD-5 is to colibacillary fungistatic effect figure.
Wherein, the penbritin (Amp) of 1 and 2 expression 100 μ L 0.02mg/mL; The fermented liquid supernatant liquid of 3 and 4 expression 100 μ L recombination yeast S78/pVT102U-α-HD5.
Embodiment
The structure of the cloning and expressing carrier of embodiment 1, phylaxin gene
1, NCBI finds the protein sequence of HD5, with reference to the codon preference of large S. cervisiae, design the DNA sequence dna of its codified.
2, design bridging primer, and hold introducing restriction enzyme site and corresponding protection base at the 5 ' end and 3 ' of sequence.
Bridging inner primer:
Upstream primer HD5-F1:
5 '-TAAGAGATGTTATTGTAGAACTGGTAGATGTGCTACTAGAGAATCATTGTCTGGTG TCT-3 ', is shown in SEQ ID NO:1,
Downstream primer HD5-R1:
5 '-TTATCTACAACACAATCTATACAATCTACCACTAATTTCACAGACACCAGACAATG ATT-3 ', is shown in SEQ ID NO:2;
Bridging Outside primer:
Upstream primer HD5-F2:5 '-CG
tCTAGAtAAGAGATGTTA-3 ' (XbaI), is shown in SEQ ID NO:3,
Downstream primer HD5-R2:5 '-CCG
aAGCTTtATCTACAACA-3 ' (HindIII), is shown in SEQ ID NO:4.
3, the acquisition of phylaxin gene
PCR reaction system is built with reference to Thermo company Pfu DNA Polymerase specification sheets.First round PCR reaction system is: the Pfu Buffer (10 ×) of 2.5 μ L, the MgSO of 2.0 μ L
4(25mM), the dNTPs (2.5uM) of 2.0 μ L, the HD5-F1 (10pmol/ μ L) of 1.0 μ L, the HD5-R1 (10pmol/ μ L) of 1.0 μ L, the ddH of 16 μ L
2the Pfu DNA Polymerase (2.5U/ μ L) of O and 0.5 μ L.Reaction process is: 95 DEG C of denaturation 1min; 94 DEG C of sex change 30sec, 68 DEG C of annealing 30sec, 68 DEG C extend 30sec, circulate 25 times; 68 DEG C of whole ends extend 5min.Second takes turns PCR reaction system is: the HD5-F2 (10pmol/ μ L) of the Pfu Buffer (10 ×) of 0.5 μ L, the dNTPs (2.5uM) of 0.5 μ L, 1.0 μ L, the HD5-R2 (10pmol/ μ L) of 1.0 μ L, 20 μ L first round PCR primer and 2.0 μ L ddH2O.Reaction process is: 95 DEG C of denaturation 1min; 94 DEG C of sex change 30sec, 52 DEG C of annealing 40sec, 72 DEG C extend 40sec, circulate 35 times; 72 DEG C of whole ends extend 7min again.The HD5 phylaxin gene fragment nucleotides sequence obtained is classified as:
cGTCTAGATAAGAGA(this is XbaI restriction enzyme site and signal peptide restriction enzyme site protein sequence) TGTTATTGTAGAACTGGTAGATGTGCTACTAGAGAATCATTGTCTGGTGTCTGTGA AATTAGTGGTAGATTGTATAGATTGTGTTGTAGATA
aAGCTTcGG (this is HindIII restriction enzyme site) (SEQ ID NO:5), the protein amino acid sequence of its correspondence is CYCRTGRCATRESLSGVCEISGRLYRLCCR (SEQ IDNO:6); PCR primer 3% agarose gel electrophoresis is observed, and see the amplified band (Fig. 1) of about 100bp, showing increases obtains target product.
4, the structure of recombinant expression vector
HD5 phylaxin gene fragment process 3 obtained uses glue recovery method to reclaim, and reclaims product XbaI and HindIII and carries out double digestion process, imitate extracting and purifying, add Virahol subzero treatment and be precipitated thing, dissolve after drying precipitate with ddH2O with phenol; Same method carries out double digestion process to secretor type saccharomyces cerevisiae expression pVT102U-α plasmid (Fig. 2), and glue reclaims the fragment of about 6.9kb.After the pVT102U-α plasmid mixing after the HD5 gene fragment after 3 μ L double digestions and 5 μ L double digestions, add 1 μ LT4DNA ligase enzyme Buffer (10 ×) and 1 μ L T4DNA ligase enzyme.16 DEG C connect 12h, will connect product conversion to DH5 α competence, and the LB flat board containing penbritin cultivates 12h for 37 DEG C; PVT102U-α plasmid is preserved by Agricultural University Of Hunan's Cell Biology Experiment room.
5, the qualification of positive bacterium colony
On the flat board containing single bacterium colony obtained from process 4, the several single bacterium colony of picking is dissolved in 20 μ L ddH2O respectively, carries out bacterium colony PCR reaction as template.Reaction system is: the Taq enzyme (2.5U/ μ L) of the dNTPs (2.5uM) of the template bacterium liquid of 5.0 μ L, the Taq Buffer (10 ×) of 2.5 μ L, 2.0 μ L, the HD5-F2 (10pmol/ μ L) of 1.0 μ L, the HD5-R2 (10pmol/ μ L) of 1.0 μ L, the ddH2O of 13 μ L and 0.5 μ L.Reaction process is: 95 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 58 DEG C of annealing 40sec, 72 DEG C extend 40sec, circulate 35 times; 72 DEG C of whole ends extend 5min again.Reaction terminates to carry out 1% agarose gel electrophoresis observation (Fig. 3) to PCR primer afterwards, and known mono-clonal bacterium colony contains goal gene fragment.
By the single bacterium colony solution inoculum containing goal gene fragment to containing enlarged culturing 12h in the LB liquid nutrient medium of penbritin, preserve a bacterial classification, residue is extracted plasmid and is delivered to biotech firm's order-checking, the plasmid called after pVT102-α-HD5 that sequencing result is correct.
The conversion of embodiment 2, recombinant Saccharomyces cerevisiae S78 expression vector
1, alkaline lysis extracts recombinant Saccharomyces cerevisiae S78 expression vector pVT102-α-HD5 plasmid in a large number.
2, electroporated
1) Host Strains: S78
YPD substratum: yeast extract paste 10g, peptone 20g, glucose 20g, agar 15g, pH 7.0.
YSD substratum: yeast nitrogen basis 6.7g, glucose 20g, leucine 200mg, VITAMIN B4 100mg, inositol 200mg, agar 15g, pH 7.0.
1M sorbyl alcohol: 182.17g sorbyl alcohol is dissolved in 1L ddH2O.
2) yeast saccharomyces cerevisiae electricity transforms
1. strain inoculation is in YPD culture medium flat plate, cultivates 2-3 days for 30 DEG C.
2. choose single colony inoculation to containing in the triangular flask of liquid YPD medium, cultivate 16h with 150r/min concussion for 30 DEG C.Be placed in 4 DEG C of standing 15min.
3. get 1.4mL bacterium liquid to manage to EP, with the centrifugal 5min of 6000r/min, abandon supernatant for 4 DEG C, collect thalline.
4. bacterium is resuspended in the deionized water of 800 μ L precoolings.With the centrifugal 5min of 6000r/min, abandon supernatant for 4 DEG C, collect thalline.
5. bacterium is resuspended in the deionized water of 500 μ L precoolings.With the centrifugal 5min of 6000r/min, abandon supernatant for 4 DEG C, collect thalline.
6. bacterium is resuspended in the sorbyl alcohol of the 1mol/L of 200 μ L precoolings.With the centrifugal 5min of 6000r/min, abandon supernatant for 4 DEG C, collect thalline.
7. bacterium is resuspended in the sorbyl alcohol of the 1mol/L of 100 μ L precoolings.Add 1ng DNA, altogether ice bath 30min.
8. ice bath bacterium liquid puts into electric shock cup altogether, and 1700V 5ms shocks by electricity.
9. mix with the sorbyl alcohol of 800 μ L YPD substratum and 200 μ L 1mol/L, wash out thalline in electric shock cup, do not shake renewal cultivation 5h.
10. by the centrifugal 5min of bacterium liquid 6000r/min after renewal cultivation, remove 800 μ L supernatants, will remain supernatant and thalline mixes, then be coated with on YSD solid plate, be inverted for 30 DEG C and cultivate 3-4 days, the transformant being grown on YSD substratum is positive transformant.
Detection of expression in yeast saccharomyces cerevisiae of embodiment 3, HD5 and separation and purification
1, on picking YSD flat board, diameter is 0.5cm bacterial plaque enlarged culturing 12h in 10mL YSD solution, then proceeds in 50mLYSD solution with the ratio of l:25,30 DEG C, 200r/min, cultivates 12h; Finally proceed in 1L YPD solution with the ratio of 1:25,30 DEG C, 200r/min, enlarged culturing 3-4 days.Removing thalline, collects in fermented liquid supernatant, to carry out detection of expression and separation and purification.
2, agar diffusion method detects and Determination of biological activity alexinic expression
After the fermented liquid supernatant centrifugal segregation precipitation in process 1, temporary for subsequent use with-80 DEG C of refrigerators.Choose the single bacterium colony of streptococcus aureus and intestinal bacteria to be connected in liquid nutrient medium 37 DEG C and to cultivate 12h, add the solid medium being cooled to about 50 DEG C and shake up rear bed board.Punch on flat board with aseptic punch tool after culture medium solidifying, drip 100 μ L fermented liquid supernatant liquid for subsequent use in hole, positive control is done with the Amp microbiotic of 100 μ L0.02mg/mL, in conjunction with see Fig. 4 and Fig. 5, known, fermented liquid supernatant has bacteriostatic activity, and to Gram-negative bacteria (intestinal bacteria) and gram-positive microorganism (streptococcus aureus) all effective, show to have the HD-5 of biologic activity successful expression secreting in fermented liquid.
Claims (4)
1. the method for a human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae, it is characterized in that, the method utilizes secretor type saccharomyces cerevisiae expression to build the recombinant Saccharomyces cerevisiae expression vector containing human α-defensin 5 gene, the recombinant Saccharomyces cerevisiae expression vector built is imported in yeast saccharomyces cerevisiae, obtain recombinant Saccharomyces cerevisiae, cultivate recombinant Saccharomyces cerevisiae and human α-defensin 5 gene is expressed.
2. the method for human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae as claimed in claim 1, it is characterized in that, described secretor type saccharomyces cerevisiae expression is pVT102U-α plasmid.
3. the method for human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae as claimed in claim 2, it is characterized in that, described recombinant Saccharomyces cerevisiae expression vector is pVT102-α-HD5.
4. the method for human α-defensin 5 secreting, expressing in yeast saccharomyces cerevisiae according to any one of claim 1-3, is characterized in that, the described method imported in yeast saccharomyces cerevisiae by the recombinant Saccharomyces cerevisiae expression vector of structure is electroporated method.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110804091A (en) * | 2019-10-18 | 2020-02-18 | 中国人民解放军陆军军医大学 | Human intestinal defensin 5 derived linear polypeptide and preparation method and application thereof |
| CN113999870A (en) * | 2020-02-26 | 2022-02-01 | 森瑞斯生物科技(深圳)有限公司 | Recombinant saccharomyces cerevisiae for expressing CBDAS and construction method and application thereof |
| CN115558613A (en) * | 2022-08-17 | 2023-01-03 | 江苏亢钧生物科技有限公司 | Culture medium for improving expression efficiency of inducing king cobra antimicrobial peptide OH-CATH30 and preparation method thereof |
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2015
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110804091A (en) * | 2019-10-18 | 2020-02-18 | 中国人民解放军陆军军医大学 | Human intestinal defensin 5 derived linear polypeptide and preparation method and application thereof |
| CN110804091B (en) * | 2019-10-18 | 2022-07-12 | 中国人民解放军陆军军医大学 | Human intestinal defensin 5 derived linear polypeptide and preparation method and application thereof |
| CN113999870A (en) * | 2020-02-26 | 2022-02-01 | 森瑞斯生物科技(深圳)有限公司 | Recombinant saccharomyces cerevisiae for expressing CBDAS and construction method and application thereof |
| CN113999870B (en) * | 2020-02-26 | 2024-02-20 | 森瑞斯生物科技(深圳)有限公司 | Recombinant saccharomyces cerevisiae for expressing CBDAS, and construction method and application thereof |
| CN115558613A (en) * | 2022-08-17 | 2023-01-03 | 江苏亢钧生物科技有限公司 | Culture medium for improving expression efficiency of inducing king cobra antimicrobial peptide OH-CATH30 and preparation method thereof |
| CN115558613B (en) * | 2022-08-17 | 2024-04-09 | 江苏亢钧生物科技有限公司 | Culture medium for improving expression efficiency of induced cobra antibacterial peptide OH-CATH30 and preparation method thereof |
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