CN103205477A - Method for producing recombinant duck-beta-defensin-2 protein - Google Patents
Method for producing recombinant duck-beta-defensin-2 protein Download PDFInfo
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- CN103205477A CN103205477A CN201310056859XA CN201310056859A CN103205477A CN 103205477 A CN103205477 A CN 103205477A CN 201310056859X A CN201310056859X A CN 201310056859XA CN 201310056859 A CN201310056859 A CN 201310056859A CN 103205477 A CN103205477 A CN 103205477A
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- alexin
- yeast
- avbd2
- duck
- beta
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Abstract
A method for producing a recombinant duck-beta-defensin-2 protein. The method is characterized by comprising an in vitro inducible expression process of a yeast recombinant converter containing duck beta-defensin-2. The process is as below: (1) induced expression of a positive yeast converter. Results show that mycelium protein subjected to induced culture has a quite obvious expression band of 4.32kD compared with a control group, which is not induced; through efficient liquid chromatography-tandem mass spectrometry identification, the mycelium protein has an amino acid sequence consistent with that of an objective protein; therefore, the target gene has been expressed, and the recombinant duck beta--defensin-2 protein has been produced. Compared with the prior art, the invention has advantages of antibiosis, growth activity promoting, suitability for application in animal production, low cost and high production efficiency.
Description
Technical field
The present invention relates to the production method of the gene in a kind of animal organism pharmaceutical engineering field.
Background technology
The feeding animal of modern livestock industry is when having highly yielding ability, and is responsive further to environmental stress and cause of disease.Particularly under the intensive farm condition, feeding animal face more stress with the invasion and attack of cause of disease.During producing, herding presses for immunopotentiating agent and the growth stimulant of a kind of efficient, toxicological harmless, noresidue, when improving feeding animal disease resistant power, can improve the production performance of feeding animal and the safety of animal product again, to promote the sound development of livestock industry, reduce herding production to the destruction of human health and ecotope.Forefathers discover, alexin has the active and superpower anti-microbial effect of broad-spectrum sterilization, and the mechanism of action is special, microorganism is difficult for producing resistance, tumour cell also had cytotoxic effect, also can be used as simultaneously immunostimulant and be used for regulating the animal body immunity system, represented a new way of developing desirable promotes growth and immunological enhancement to people.
Because the natural alexin content of animal body is low, directly extraction far can not be satisfied the demand, and the cost height, yield is low, operation is numerous; Chemosynthesis is feasible but cost is too high, uses and is limited to; Along with the development of genetic engineering technique, by gene recombination technology production alexin polypeptide, become possibility with the needs that satisfy research and produce.At present, alexinic genetically engineered has been the focus of biomedical sector research.Have extraordinary application prospect based on alexinic important physiological function and in production reality, we have carried out a kind of invention research of the duck beta-alexin-2 albumen in-vitro recombination expression production method of recombinating.
Summary of the invention
Goal of the invention of the present invention is to provide a kind of antibiotic growth-promoting activity, is suitable in herding is produced, using, and the production method of a kind of duck beta-alexin-2 albumen of recombinating that production cost is low, production efficiency is high.
The present invention's duck beta-alexin-2 gene of recombinating is to realize like this, comprise successively the design of the gene nucleotide series of duck beta-alexin-2(AvBD2) and external synthetic, recombinant plasmid pPICZ alpha-A-AvBD2 building process, contain yeast recombinant conversion of duck beta-alexin-2(AvBD2) screening process, contain the outer abduction delivering process of yeast recombinant conversion daughter of duck beta-alexin-2(AvBD2)
Duck beta-alexin of the present invention-2(AvBD2) the gene nucleotide series design is achieved in that with external building-up process
With reference to the duck AvBD2 gene cDNA gene order of registering among the GenBank, according to the yeast codon preference duck AvBD2 gene mature peptide section nucleotide sequence is transformed simultaneously, utilize method for synthesizing gene synthetic (serve sea give birth to worker's biotechnology company limited to carry out gene synthetic) then, transform the back synthetic the duck beta-alexin-2(AvBD2) gene nucleotide series is as follows:
GATATGTTTTTGTGTAGAATTGGTTCTTGTCATTTTGGTAGATGTCCAATTCATTTGGTTAGAGTTGGTTCCTGTTTTGGTTTTAGATCTTGTTGTAAGTCTCCATGGGATGTTTAA。
Recombinant plasmid pPICZ alpha-the A-AvBD2 that contains reorganization duck beta-alexin-2 gene of the present invention is achieved in that
The building process of recombinant plasmid pPICZ alpha-A-AvBD2 is successively:
1, the setting up procedure of upstream region of gene primer, downstream primer of duck beta-alexin-2(AvBD2): the duck beta-alexin-2(AvBD2) gene nucleotide series designs behind multiple comparisons according to synthetic, the upstream primer design is at this gene initiating terminal, according to the restriction enzyme site on expression vector pPICZ α-A, added during the upstream primer design simultaneously
XhoI restriction enzyme site and Kex2 and Ste13 protease cracking site,
XhoBe added with 3 protectiveness base TCT before the I restriction enzyme site; Downstream primer designs at the gene end, and adds
NotThe I restriction enzyme site,
NotBe added with 4 protectiveness bases G CTG before the I restriction enzyme site site, set duck beta-alexin-2(AvBD2) upstream region of gene primer, downstream primer are respectively,
The upstream region of gene primer of duck beta-alexin-2(AvBD2):
5 '-TCT
CTCGAGAAAAGAGAGGCTGGATATGTTTTTGTGTAG-3 ' Xho I restriction enzyme site,
The gene downstream primer of duck beta-alexin-2(AvBD2):
5 '-GCTG
GCGGCCGCTTAAACATCCCAT-3 ' Not I restriction enzyme site,
2, the pcr amplification process of gene fragment of duck beta-alexin-2(AvBD2): the duck beta-alexin-2(AvBD2) gene is template with synthetic, add the ExTaq archaeal dna polymerase, on the duck beta-alexin-2(AvBD2)/downstream primer, dNTPs carries out amplified reaction, the reaction system of PCR is: 10 * PCR Buffer(comprises 0.5mmol/L MgCl2,50mmol/L KCl, 10mmol/L Tris(Tutofusin tris) HCl, 0.001% gelatin) 5 μ L, 4 * dNTPs(comprises dATP(triphosphoric acid adenyl-deoxyribonucleotide), dTTP(triphosphoric acid thymidylic acid), dCTP(triphosphoric acid deoxycytidylic acid) and the acid of dGTP(triphosphoric acid guanine deoxyribonucleoside) each 2.5mmol/L) 4 μ L, concentration is on the duck beta-alexin of 20 μ mol/L-2(AvBD2), each 1 μ L of downstream primer, template is the gene 1 μ L of synthetic duck beta-alexin-2(AvBD2), the ddH2O(distilled water) 37.5 μ L, concentration is the ExTaq archaeal dna polymerase 0.5 μ L of 5 μ mol/L, and reaction process is followed successively by: a, handle 3min for 94 ℃; B, successively 94 ℃ handle 30s, 56 ℃ and handle 30s, 72 ℃ and handle 30s; Reaction process is carried out 30 circulations; C, 72 ℃ of extension 10min; Namely obtain the PCR product of gene of duck beta-alexin-2(AvBD2), the duck beta-alexin-2(AvBD2) the PCR product of gene is observed at 1.5% agarose gel electrophoresis, and the amplified band of the about 130bp of visible size is consistent with expected result.
3, the structure of recombinant plasmid pPICZ alpha-A-AvBD2: with the PCR product of the gene of the duck beta-alexin of process 2-2(AvBD2) with atmosphere chloroform extracting and purifying, add ethanol and obtain throw out, to include 10mmol/L TrisHCl with TE(TE after the drying precipitate, 1mmol/L EDTA(ethylenediamine tetraacetic acid (EDTA))) dissolving, the TE solute is used
XohI,
NotI carries out double digestion to be handled, and glue reclaims the band of the about 130bp of size; With same method pPICZ α-A plasmid being carried out double digestion handles, glue reclaims the dna fragmentation of the about 3.6kb of size, get pPICZ α-A plasmid that glue reclaims behind the gene fragment of the duck beta-alexin that glue reclaims behind the 5 μ L double digestions-2(AvBD2) and the 3 μ L double digestions respectively, 10 * T4 the DNA that adds 1 μ L connects Buffer(10 * T4 DNA connection Buffer and includes 0.5mol/L TrisHCl, 0.1mol/L MgCl2,50mmol/L DTT(dithiothreitol (DTT)), 5mmol/L DATP, 0.25mg/mL the BSA(bovine serum albumin)), 1 μ L T4 dna ligase, connect processing 18 hours down at 16 ℃, to connect product and transform Top 10 competent cells, in containing the less salt LB agar culture plate of 25 μ g/mL Zeocin, 37 ℃ of overnight incubation are namely finished the structure of recombinant plasmid pPICZ alpha-A-AvBD2.White colony longer on the culture plate is the positive bacteria that contains recombinant plasmid pPICZ alpha-A-AvBD2.PPICZ α-A plasmid is the product of Invitrogen company.
The production method of the recombination yeast positive transformant of Fa Ming reorganization duck beta-alexin-2 albumen is not achieved in that
1, the preparation of recombinant plasmid and linearizing: Top 10 positive bacterias that picking is transformed into recombinant plasmid pPICZ alpha-A-AvBD2 are inoculated in 25 mL and contain in the less salt liquid LB substratum of 25 μ g/mL Zeocin, and 37 ℃, 24 h are cultivated in 220 rpm joltings.With bacterium liquid in 4 ℃, centrifugal 10 min of 5 000rpm, precipitum.Use plasmid DNA extraction agent box extracting plasmid then.With
SacThe I enzyme is with the recombinant plasmid pPICZ alpha-A-AvBD2 linearizing of extracting.
2, the preparation of pichia spp electricity transformed competence colibacillus cell: inoculate the single bacterium colony of pichia spp host bacterium X33 and contain the 1wt% yeast extract to 5mL YPD(, 21wt % peptone, 21wt % glucose) in the substratum, 30 ℃ of incubated overnight.Inoculate 0.1～0.5mL incubated overnight bacterium liquid then to the fresh YPD nutrient solution of 500mL, 30 ℃ of cultivations make OD600nm reach 1.2～1.3.4 ℃ of 3 centrifugal 5min of 000r/m abandons supernatant; Successively with the resuspended thalline washing of the ice-cold 1mol/L Sorbitol Powder of 500mL, 250mL ice bath aqua sterilisa and 20mL, 4 ℃ of 3 centrifugal 5min of 000r/m, centrifugal 3 times; At last cell is resuspended in the 1mol/L Sorbitol Powder of 1mL ice bath sterilization, is the pichia spp competent cell.
3, the electric shock of pichia spp transforms: get the ready pichia spp competent cell of 80 μ L, mix with the linearizing recombinant plasmid pPICZ alpha-A-AvBD2 of 5～10 μ g, transfer in the 0.2cm electricity revolving cup of ice bath, ice bath 15min, in the conversion of shocking by electricity of 2000-Y type gene introducing apparatus, electric pulse parameter is voltage 1500V, electrical capacity 25 μ F, resistance 200 Ω.The 1mol/L Sorbitol Powder that adds the 1mL ice bath after the electric shock immediately transforms in the cuvette to electricity, suspension in the cuvette is transferred in the test tube of 15mL of a sterilization, test tube was hatched under 30 ℃ 1～2 hour, get 10,25,50,100 are taped against respectively and contain 200 μ g/mL Zeocin YPDS(1% yeast extracts with 200uL, 2% peptone, 2% glucose, the 1M Sorbitol Powder, the 20g agar powder) on the flat board screening high copy transformant, 30 ℃ of dull and stereotyped 2 ~ 3d of hatching are yeast recombinant conversion that contains duck beta-alexin-2(AvBD2) to milky yeast conversion bacterium colony occurring.
4, the PCR of pichia spp transformant identifies: extracting contains yeast recombinant conversion of duck beta-alexin-2(AvBD2), be template with it, add the ExTaq archaeal dna polymerase, on the duck beta-alexin-2(AvBD2)/downstream primer, dNTPs carries out amplified reaction, the reaction system of PCR is: 10 * PCR Buffer(comprises 0.5mmol/L MgCl2,50mmol/L KCl, 10mmol/L TrisHCl, the 0.001wt% gelatin) 5 μ L, 4 * dNTPs(comprises dATP, dTTP, dCTP, each 2.5mmol/L of dGTP) 4 μ L, concentration is on the duck AvBD2 of 20 μ mol/L, each 1 μ L of downstream primer, template is the pastoris genomic dna 1 μ L that extracting transforms, the ddH2O(distilled water) 37.5 μ L, concentration is the ExTaq archaeal dna polymerase 0.5 μ L of 5 μ mol/L, reaction process is followed successively by: a, handle 3min for 94 ℃; B, successively 94 ℃ handle 30s, 56 ℃ and handle 30s, 72 ℃ and handle 30s; Reaction process is carried out 30 circulations; C, 72 ℃ of extension 10min; Pcr amplification product is observed at the 1.5wt% agarose gel electrophoresis, and the visible amplified band of about 130bp namely shows the positive yeast transformant of yeast of institute's extracting.
The recombinate production method of duck beta-alexin-2 albumen of the present invention is achieved in that
1, the abduction delivering of positive yeast transformant: the positive yeast transformant of picking through identifying is inoculated in the 3 mL YPD liquid 30 ℃, 250 r/min jolting overnight incubation; Be inoculated in 50 mL BMGY(1wt% yeast extracts, 2 wt % peptones, 1mol/L potassium phosphate buffer (pH6.0), 1.34 wt %YNB(does not have yeast nitrogen), 4 * 10-5 wt % vitamin H, 1 wt % glycerine) in the body substratum, 30 ℃ of shaking culture 24 h, reach 2～6 o'clock centrifugal supernatant discarded to thalline OD600nm, add 25 mL BMMY liquid nutrient mediums (1 wt % yeast extract, 2 wt % peptones, 1mol/L potassium phosphate buffer (pH6.0), 1.34 wt %YNB(does not have yeast nitrogen), 4 * 10-5 wt % vitamin H, 0.5 wt % methyl alcohol), continuous induction is cultivated 72 h, in inducing process, every interval 24h adds 100 wt% methyl alcohol, and to make its final concentration be 0.5%, is cultured to 72h and collects sample, centrifugal, get supernatant and carry out the Tricine-SDS-PAGE electrophoresis, the result shows that the tropina behind the inducing culture compares with the control group of not inducing, and it is very significantly protein expression band of 4.32kD that a size is arranged.Identify through Ultra Performance Liquid Chromatography-esi-msn, match with the target protein aminoacid sequence, show that goal gene has obtained expressing duck beta-alexin-2 gene protein of namely recombinating and produced.
To contain fermented liquid that the outer abduction delivering process of yeast recombinant conversion daughter of duck beta-alexin-2(AvBD2) produces is directly used in the in-vitro antibacterial experiment and shows the reorganization duck beta-alexin-2(AvBD2) albumen has Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas, streptococcus aureus, subtilis ability.The interior therapeutic experiment finds that beta-alexin-2(AvBD2) albumen has vivo control intestinal bacteria and Salmonellas action effect preferably to the reorganization duck.
The present invention compared with the prior art, be that reorganization duck AvBD2 albumen is carried out vivoexpression production research, adopt the yeast codon optimization method that duck AvBD2 gene nucleotide is optimized, be conducive to the abduction delivering production of duck AvBD2 in yeast, can improve expression amount.Be exactly in addition, yeast belongs to eukaryotic expression system, and the recombinant protein of expressing is conducive to the maintenance of recombinant protein activity more close to native protein.Detect the recombinant protein of also expressing as can be seen from activity and present the antibiotic growth-promoting activity that duck AvBD2 albumen has, be suitable in herding is produced, using, and production cost is low, production efficiency is high.
Embodiment:
Now in conjunction with the embodiments the present invention is described in further detail:
Duck beta-alexin of the present invention-2(AvBD2) the gene nucleotide series design is achieved in that with external building-up process
With reference to the duck AvBD2 gene cDNA gene order of registering among the GenBank, according to the yeast codon preference duck AvBD2 gene mature peptide section nucleotide sequence is transformed simultaneously, utilize method for synthesizing gene synthetic (serve sea give birth to worker's biotechnology company limited to carry out gene synthetic) then, transform the back synthetic the duck beta-alexin-2(AvBD2) gene nucleotide series is as follows:
GATATGTTTTTGTGTAGAATTGGTTCTTGTCATTTTGGTAGATGTCCAATTCATTTGGTTAGAGTTGGTTCCTGTTTTGGTTTTAGATCTTGTTGTAAGTCTCCATGGGATGTTTAA。
Recombinant plasmid pPICZ alpha-the A-AvBD2 that contains this gene of the present invention is achieved in that
The building process of recombinant plasmid pPICZ alpha-A-AvBD2 is successively:
1, the setting up procedure of upstream region of gene primer, downstream primer of duck beta-alexin-2(AvBD2): the duck beta-alexin-2(AvBD2) gene nucleotide series designs behind multiple comparisons according to synthetic, the upstream primer design is at this gene initiating terminal, according to the restriction enzyme site on expression vector pPICZ α-A, added during the upstream primer design simultaneously
XhoI restriction enzyme site and Kex2 and Ste13 protease cracking site,
XhoBe added with 3 protectiveness base TCT before the I restriction enzyme site; Downstream primer designs at the gene end, and adds
NotThe I restriction enzyme site,
NotBe added with 4 protectiveness bases G CTG before the I restriction enzyme site site, set duck beta-alexin-2(AvBD2) upstream region of gene primer, downstream primer are respectively,
The upstream region of gene primer of duck beta-alexin-2(AvBD2):
5 '-TCT
CTCGAGAAAAGAGAGGCTGGATATGTTTTTGTGTAG-3 ' Xho I restriction enzyme site,
The gene downstream primer of duck beta-alexin-2(AvBD2):
5 '-GCTG
GCGGCCGCTTAAACATCCCAT-3 ' Not I restriction enzyme site,
2, the pcr amplification process of gene fragment of duck beta-alexin-2(AvBD2): the duck beta-alexin-2(AvBD2) gene is template with synthetic, add the ExTaq archaeal dna polymerase, on the duck beta-alexin-2(AvBD2)/downstream primer, dNTPs carries out amplified reaction, the reaction system of PCR is: 10 * PCR Buffer(comprises 0.5mmol/L MgCl2,50mmol/L KCl, 10mmol/L TrisHCl, 0.001% gelatin) 5 μ L, 4 * dNTPs(comprises dATP, dTTP, dCTP, each 2.5mmol/L of dGTP) 4 μ L, concentration is on the duck beta-alexin of 20 μ mol/L-2(AvBD2), each 1 μ L of downstream primer, template is the gene 1 μ L of synthetic duck beta-alexin-2(AvBD2), the ddH2O(distilled water) 37.5 μ L, concentration is the ExTaq archaeal dna polymerase 0.5 μ L of 5 μ mol/L, and reaction process is followed successively by: a, handle 3min for 94 ℃; B, successively 94 ℃ handle 30s, 56 ℃ and handle 30s, 72 ℃ and handle 30s; Reaction process is carried out 30 circulations; C, 72 ℃ of extension 10min; Namely obtain the PCR product of gene of duck beta-alexin-2(AvBD2), the duck beta-alexin-2(AvBD2) the PCR product of gene is observed at 1.5% agarose gel electrophoresis, and the amplified band of the about 130bp of visible size is consistent with expected result.
3, the structure of recombinant plasmid pPICZ alpha-A-AvBD2: with the PCR product of the gene of the duck beta-alexin of process 2-2(AvBD2) with atmosphere chloroform extracting and purifying, add ethanol and obtain throw out, to include 10mmol/L TrisHCl with TE(TE after the drying precipitate, 1mmol/L EDTA) dissolving, the TE solute is used
XohI,
NotI carries out double digestion to be handled, and glue reclaims the band of the about 130bp of size; With same method pPICZ α-A plasmid being carried out double digestion handles, glue reclaims the dna fragmentation of the about 3.6kb of size, get pPICZ α-A plasmid that glue reclaims behind the gene fragment of the duck beta-alexin that glue reclaims behind the 5 μ L double digestions-2(AvBD2) and the 3 μ L double digestions respectively, 10 * T4 the DNA that adds 1 μ L connects Buffer(10 * T4 DNA connection Buffer and includes 0.5mol/L TrisHCl, 0.1mol/L MgCl2,50mmol/L DTT, 5mmol/L DATP, 0.25mg/mL BSA), 1 μ L T4 dna ligase, connect processing 18 hours down at 16 ℃, to connect product and transform Top 10 competent cells, in containing the less salt LB agar culture plate of 25 μ g/mL Zeocin, 37 ℃ of overnight incubation are namely finished the structure of recombinant plasmid pPICZ alpha-A-AvBD2.White colony longer on the culture plate is the positive bacteria that contains recombinant plasmid pPICZ alpha-A-AvBD2.PPICZ α-A plasmid is the product of Invitrogen company.
Recombination yeast positive transformant for the production of reorganization duck beta-alexin-2 albumen is achieved in that
1, the preparation of recombinant plasmid and linearizing: Top 10 positive bacterias that picking is transformed into recombinant plasmid pPICZ alpha-A-AvBD2 are inoculated in 25 mL and contain in the less salt liquid LB substratum of 25 μ g/mL Zeocin, and 37 ℃, 24 h are cultivated in 220 rpm joltings.With bacterium liquid in 4 ℃, centrifugal 10 min of 5 000rpm, precipitum.Use plasmid DNA extraction agent box extracting plasmid then.With
SacThe I enzyme is with the recombinant plasmid pPICZ alpha-A-AvBD2 linearizing of extracting.
2, the preparation of pichia spp electricity transformed competence colibacillus cell: inoculate the single bacterium colony of pichia spp host bacterium X33 and contain the 1wt% yeast extract to 5mL YPD(, 21wt % peptone, 21wt % glucose) in the substratum, 30 ℃ of incubated overnight.Inoculate 0.1～0.5mL incubated overnight bacterium liquid then to the fresh YPD nutrient solution of 500mL, 30 ℃ of cultivations make OD600nm reach 1.2～1.3.4 ℃ of 3 centrifugal 5min of 000r/m abandons supernatant; Successively with the resuspended thalline washing of the ice-cold 1mol/L Sorbitol Powder of 500mL, 250mL ice bath aqua sterilisa and 20mL, 4 ℃ of 3 centrifugal 5min of 000r/m, centrifugal 3 times; At last cell is resuspended in the 1mol/L Sorbitol Powder of 1mL ice bath sterilization, is the pichia spp competent cell.
3, the electric shock of pichia spp transforms: get the ready pichia spp competent cell of 80 μ L, mix with the linearizing recombinant plasmid pPICZ alpha-A-AvBD2 of 5～10 μ g, transfer in the 0.2cm electricity revolving cup of ice bath, ice bath 15min, in the conversion of shocking by electricity of 2000-Y type gene introducing apparatus, electric pulse parameter is voltage 1500V, electrical capacity 25 μ F, resistance 200 Ω.The 1mol/L Sorbitol Powder that adds the 1mL ice bath after the electric shock immediately transforms in the cuvette to electricity, suspension in the cuvette is transferred in the test tube of 15mL of a sterilization, test tube was hatched under 30 ℃ 1～2 hour, get 10,25,50,100 are taped against respectively and contain 200 μ g/mL Zeocin YPDS(1% yeast extracts with 200uL, 2% peptone, 2% glucose, the 1M Sorbitol Powder, the 20g agar powder) on the flat board screening high copy transformant, 30 ℃ of dull and stereotyped 2 ~ 3d of hatching are yeast recombinant conversion that contains duck beta-alexin-2(AvBD2) to milky yeast conversion bacterium colony occurring.
4, the PCR of pichia spp transformant identifies: extracting contains yeast recombinant conversion of duck beta-alexin-2(AvBD2), be template with it, add the ExTaq archaeal dna polymerase, on the duck beta-alexin-2(AvBD2)/downstream primer, dNTPs carries out amplified reaction, the reaction system of PCR is: 10 * PCR Buffer(comprises 0.5mmol/L MgCl2,50mmol/L KCl, 10mmol/L TrisHCl, the 0.001wt% gelatin) 5 μ L, 4 * dNTPs(comprises dATP, dTTP, dCTP, each 2.5mmol/L of dGTP) 4 μ L, concentration is on the duck AvBD2 of 20 μ mol/L, each 1 μ L of downstream primer, template is the pastoris genomic dna 1 μ L that extracting transforms, the ddH2O(distilled water) 37.5 μ L, concentration is the ExTaq archaeal dna polymerase 0.5 μ L of 5 μ mol/L, reaction process is followed successively by: a, handle 3min for 94 ℃; B, successively 94 ℃ handle 30s, 56 ℃ and handle 30s, 72 ℃ and handle 30s; Reaction process is carried out 30 circulations; C, 72 ℃ of extension 10min; Pcr amplification product is observed at the 1.5wt% agarose gel electrophoresis, and the visible amplified band of about 130bp namely shows the positive yeast transformant of yeast of institute's extracting.
The production method of reorganization duck beta-alexin-2 albumen,
1, the abduction delivering of positive yeast transformant: the positive yeast transformant of picking through identifying is inoculated in the 3 mL YPD liquid 30 ℃, 250 r/min jolting overnight incubation; Be inoculated in 50 mL BMGY(1wt% yeast extracts, 2 wt % peptones, 1mol/L potassium phosphate buffer (pH6.0), 1.34 wt %YNB(does not have yeast nitrogen), 4 * 10-5 wt % vitamin H, 1 wt % glycerine) in the body substratum, 30 ℃ of shaking culture 24 h, reach 2～6 o'clock centrifugal supernatant discarded to thalline OD600nm, add 25 mL BMMY liquid nutrient mediums (1 wt % yeast extract, 2 wt % peptones, 1mol/L potassium phosphate buffer (pH6.0), 1.34 wt %YNB(does not have yeast nitrogen), 4 * 10-5 wt % vitamin H, 0.5 wt % methyl alcohol), continuous induction is cultivated 72 h, in inducing process, every interval 24h adds 100 wt% methyl alcohol, and to make its final concentration be 0.5%, is cultured to 72h and collects sample, centrifugal, get supernatant and carry out the Tricine-SDS-PAGE electrophoresis, the result shows that the tropina behind the inducing culture compares with the control group of not inducing, and it is very significantly protein expression band of 4.32kD that a size is arranged.Identify through Ultra Performance Liquid Chromatography-esi-msn, match with the target protein aminoacid sequence, show that goal gene has obtained expressing duck beta-alexin-2 gene protein of namely recombinating and produced.
Restriction enzyme Xho I used in the present invention, Not I, Sac I, T4DNA ligase enzyme, ExTaq archaeal dna polymerase etc. are all available from precious Tyke, Guangzhou bio tech ltd.Strain X-33, Zeocin microbiotic, pPICZ α-A plasmid are the product of Invitrogen company.
Antibacterial activity in vitro detect to find that reorganization duck AvBD2 albumen has Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas, streptococcus aureus, subtilis ability, and the minimal inhibitory concentrations of intestinal bacteria 44103, Salmonella choleraesuls (ATCC13312), streptococcus aureus (ATCC6538), subtilis (ATCC6633), strain isolated intestinal bacteria and strain isolated Salmonella choleraesuls is respectively 16.41,65.63,32.81,16.41,65.63 and 131.25 μ g/ml.Chick with two ages in week is object, preliminary study the effect of reorganization duck AvBD2 albumen control chick infectation of bacteria.In treatment experiment, it is best that the chick that intestinal bacteria are attacked poison is injected 250 μ g reorganization duck AvBD2 protein for treatment effect, one week back chick survival rate reach 90%; Salmonellas is attacked malicious chick, and to inject 300 μ g reorganization duck AvBD2 protein for treatment effect best, one week back chick survival rate reach 80%; The result shows that reorganization duck beta-alexin-2 albumen has vivo control effect preferably.
SEQUENCE LISTING
＜110〉Foshan Science ﹠. Technology College
Open brightness China
Korea Spro, little phoenix
Horse protects China
Finish the English assistant
Horse, quiet cloud
Thank to green plum
Lee, occasion
＜120〉a kind of production method of duck beta-alexin-2 albumen of recombinating
<130> 2011
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 117
<212> DNA
＜213〉synthetic duck alexin-2 gene
<220>
<221> CDS
<222> (1)..(117)
<400> 1
gat atg ttt ttg tgt aga att ggt tct tgt cat ttt ggt aga tgt cca 48
Asp Met Phe Leu Cys Arg Ile Gly Ser Cys His Phe Gly Arg Cys Pro
1 5 10 15
att cat ttg gtt aga gtt ggt tcc tgt ttt ggt ttt aga tct tgt tgt 96
Ile His Leu Val Arg Val Gly Ser Cys Phe Gly Phe Arg Ser Cys Cys
20 25 30
aag tct cca tgg gat gtt taa 117
Lys Ser Pro Trp Asp Val
35
<210> 2
<211> 38
<212> PRT
＜213〉synthetic duck alexin-2 gene
<400> 2
Asp Met Phe Leu Cys Arg Ile Gly Ser Cys His Phe Gly Arg Cys Pro
1 5 10 15
Ile His Leu Val Arg Val Gly Ser Cys Phe Gly Phe Arg Ser Cys Cys
20 25 30
Lys Ser Pro Trp Asp Val
35
<210> 3
<211> 39
<212> DNA
＜213〉composition sequence
<220>
<221> misc_difference
<222> (1)..(39)
<400> 3
tctctcgaga aaagagaggc tggatatgtt tttgtgtag 39
<210> 4
<211> 25
<212> DNA
＜213〉composition sequence
<220>
<221> misc_difference
<222> (1)..(25)
<400> 4
gctggcggcc gcttaaacat cccat 25
Claims (1)
1. the production method of duck beta-alexin-2 albumen of recombinating is characterized in that,
The outer abduction delivering process of yeast recombinant conversion daughter that contains duck beta-alexin-2:
(1) abduction delivering of positive yeast transformant: the positive yeast transformant of picking through identifying is inoculated in the 3 mL YPD liquid 30 ℃, 250 r/min jolting overnight incubation; Be inoculated in the 50 mL BMGY body substratum, BMGY comprises the 1wt% yeast extract, 2 wt % peptones, the pH of 1mol/L is 6.0 potassium phosphate buffer, 1.34wt% does not have yeast nitrogen, 4 * 10-5wt % vitamin H, 1%wt glycerine, 30 ℃ of shaking culture 24 h, reach 2～6 o'clock centrifugal supernatant discarded to thalline OD600nm, add 25 mL BMMY liquid nutrient mediums, the BMMY liquid nutrient medium comprises the 1wt% yeast extract, 2 wt % peptones, the pH of 1mol/L is 6.0 potassium phosphate buffer, 1.34 wt % does not have yeast nitrogen, 4 * 10-5 wt % vitamin H, 0.5 wt % methyl alcohol, continuous induction is cultivated 72 h, in inducing process, every interval 24h adds 100% methyl alcohol, and to make its final concentration be 0.5%, be cultured to 72h and collect sample, centrifugal, get supernatant and carry out the Tricine-SDS-PAGE electrophoresis, the result shows that the tropina behind the inducing culture compares with the control group of not inducing, it is very significantly protein expression band of 4.32kD that a size is arranged, identify through Ultra Performance Liquid Chromatography-esi-msn, match with the target protein aminoacid sequence, show that goal gene has obtained expressing duck beta-alexin-2 albumen of namely recombinating and produced.
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Non-Patent Citations (3)
| Title |
|---|
| 唐博等: "骆驼β-防御素CaBD-I cDNA的克隆及序列分析", 《畜牧兽医学报》 * |
| 张辉华等: "鸡β-防御素-1 cDNA的克隆及在毕赤酵母中的表达", 《畜牧兽医学报》 * |
| 王瑞琴等: "重组鸭β-防御素-2基因的克隆、表达和表达产物的生物学特性分析", 《中国农业科学》 * |
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| CN108642108A (en) * | 2018-05-16 | 2018-10-12 | 佛山科学技术学院 | A kind of production method of efficient -2 albumen of recombination duck beta-alexin |
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