CN106148381A

CN106148381A - A kind of method utilizing bacillus subtilis height efficient expression porcine defensin

Info

Publication number: CN106148381A
Application number: CN201610585836.1A
Authority: CN
Inventors: 苟兴华; 李钢; 邹辉琴; 张丹; 王娜; 张崟
Original assignee: Chengdu University
Current assignee: ROTA Bioengineering Co., Ltd.
Priority date: 2016-07-22
Filing date: 2016-07-22
Publication date: 2016-11-23
Anticipated expiration: 2036-07-22
Also published as: CN106148381B

Abstract

The present invention discloses a kind of method utilizing bacillus subtilis height efficient expression porcine defensin, belongs to gene engineering technology field.Method includes: the structure of (1) porcine defensin carrier and the synthesis of destination protein gene；(2) clone's gained gene and plasmid vector pHT43 are all carried out KpnI/BamH I double digestion, double digestion genetic fragment is attached and converts, obtains recombinant vector；(3) being transformed on bacillus subtilis by recombinant vector, screening obtains positive transformant；(4) positive transformant is inoculated in LB culture medium, cultivates under the conditions of 37 DEG C, to OD₆₀₀Adding derivant to concentration when reaching 0.8 is 2%, induced reaction 24h at 37 DEG C.The present invention has prepared Recombinant Swine alexin fusion protein, and the expression of porcine defensin fusion protein is high, establishes the method that bacillus subtilis secretion expresses antibacterial peptide, solves the problem such as source restriction of antibacterial peptide.

Description

A kind of method utilizing bacillus subtilis height efficient expression porcine defensin

Technical field

The invention belongs to gene engineering technology field, be specifically related to one and utilize bacillus subtilis height efficient expression pig to defend The method of element.

Background technology

In recent years, along with antibiotic is widely used in livestock and poultry cultivation, Resistant strain and the continuous increasing of multiple antibiotic resistant strain Adding so that the Drug resistance of pathogen is greatly improved, this not only creates strong influence to livestock and poultry cultivation, and gradually makes the mankind Anti-infective therapy is also absorbed in drug resistance crisis, makes food safety and human medical's health face huge potential safety hazard, antibiotic Use is faced with unprecedented stern challenge.Study and apply new additive agent technical side nontoxic, nuisanceless, without drug resistance Case substitutes conventional antibiotic care plans has become development health, safety, the inexorable trend of stockbreeding cultivation.

Alexin is the class cation activity polypeptide rich in cysteine, and its molecular weight is 2000-6000Da, by 29- 54 amino acid residue compositions, containing 6-8 conservative cysteine in molecule, are the weights of animal innate immune defense system Want ingredient.Alexin is wide expression in animal tissue cell, lodges in cell medium, in intracellular or extracellular Directly to kill the pathogenic microorganisms such as antibacterial, fungus, virus, it is that host resists the first of extraneous pathogenic microorganism invasion and prevents Line.Alexin due to its have has a broad antifungal spectrum, antibacterial mechanisms is special, be not likely to produce drug resistance, remain low, normal to animal self The features such as cell not injury, make one of alexin optimal candidate becoming substitute antibiotics, and future will have in Production of Livestock and Poultry Have broad application prospects.

Antibacterial peptide has the small peptide of broad spectrum antibiotic activity as a class, has molecular weight low, and alkalescence is strong, thermally-stabilised, water-soluble Property good and the most not to features such as eukaryotic cell work, especially there is the Antibacterial Mechanism of uniqueness, it is not likely to produce by antibacterial Drug resistance, therefore it is expected to become a new generation's antibacterial, to solve a large amount of Resistant strains using conventional antibiotic to result in This global problem.Therefore carrying out the research to pig antibacterial peptide and have most important theories and realistic meaning, pig antibacterial peptide can conduct Antistaling agent for meat food and additive for farm animal feed, therefore, obtain antibacterial Toplink by clonal expression pig antibacterial skin peptide gene and bring Social benefit and economic benefit greatly.

At present, the most the cattle in mammal, sheep, rabbit, human alpha-defensin are made some researchs, and to antibacterial peptide Research also focuses primarily upon the research of insect antimicrobial peptide, few to the research report of porcine defensin and pig antibacterial peptide, prevents about pig Drive element restructuring in bacillus subtilis and amalgamation and expression there is not yet document report, be therefore badly in need of one and can stably obtain pig The method of antibacterial peptide.

Summary of the invention

The purpose of the present invention is contemplated to solve above-mentioned technical problem, and provides one to utilize the efficient table of bacillus subtilis The method reaching porcine defensin so that porcine defensin is recombinated in bacillus subtilis, and fusion protein is at bacillus subtilis Bacterium is able to high efficient expression, it is possible to stably obtaining pig antibacterial peptide, porcine defensin expression reaches 3mg/L.

To achieve these goals, the technical solution used in the present invention is, one utilizes bacillus subtilis height efficient expression The method of porcine defensin, comprises the steps:

(1) choose porcine defensin BD gene, BD gene adds SacB gene and SamyQ gene, obtains engineered protein SS-BD gene；

(2) two ends of step (1) gained engineered protein SS-BD gene are carried out KpnI/BamH I double digestion and obtains SS-BD Genes of interest fragment；

(3) select bacillus subtilis plasmid vector pHT43 to carry out KpnI/BamH I double digestion, obtain pHT43 double digestion Genetic fragment；

(4) step (2) gains are attached with step (3) gains and convert, obtain recombinant vector pHT-BD, will Recombinant vector pHT-BD is transformed on bacillus subtilis, the positive transformant that screening can be grown on chloromycetin flat board；

(5) be inoculated in LB culture medium after the positive transformant monoclonal that will filter out, be placed in shaking table, 37 DEG C, Incubated overnight under the conditions of 180rpm, obtains one-level culture fluid, by one-level culture fluid as 1% inoculum concentration be inoculated in LB described in 50mL In culture medium, be placed in shaking table, 37 DEG C, cultivate under the conditions of 225rpm, to OD₆₀₀Adding derivant when reaching 0.8 to concentration is 2% (m/v), induced reaction 24h at 37 DEG C.

The present invention uses bacillus subtilis to be Host Strains, and selecting pHT43 is peptide expression carrier, sets up hay spore The method of bacillus secreting, expressing antibacterial peptide, solves the problems such as antibacterial peptide source restriction.Bacillus subtilis can be utilized simultaneously Probiotic bacteria character, it is possible to produce antibiotics, suppress the growth of other antibacterials, also has in the intestinal of poultry and the strongest takes oxygen energy by force Power, promotes that the aspects such as the feed conversion rate of animal, diseases prevention rate, growth of animal of digesting and assimilating, improve of raising poultry nutritive play important Effect, the production application for bacillus subtilis provides theoretical foundation.

The present invention is by the transformation to porcine defensin gene, and through KpnI/BamH I double digestion, selects plasmid further Carrier pHT43 carries out double digestion reaction, and the two is attached and converts, it is thus achieved that recombinant vector pHT-BD, by recombinant vector PHT-BD is transformed into bacillus subtilis, obtains positive transformant, selects suitable LB culture medium, by twice training after clone Support, it is thus achieved that destination protein, after testing, porcine defensin activity reaches 3mg/L.

Further, the sequence of described porcine defensin BD gene is as follows:

gaccactacatatgtgccaagaaaggggggacctgcaacttctccccctgcccgctcttcaacaggattgaagggac ctgttacagtggcaaggccaagtgctgcatccgctga。

Further, described step (2) is 5 ' end addition BamH I restriction enzyme sites at engineered protein SS-BD gene, 3 ' ends add KpnI restriction enzyme site.

The sequence of described SS-BD genes of interest is as follows:

KpnI

ggtaccccttttatgattttctatcaaacaaaagaggaaaatagaccagttgcaatccaaacgagagtctaatagaa tgaggtcgaaaagtaaatcgcgcgggtttgttactgataaagcaggcaagacctaaaatgtgtaaagggcaaagtgt atactttggcgtcaccccttacatattttaggtctttttttattgtgcgtaactaacttgccatcttcaaacaggag ggctggaagaagcagaccgctaacacagtacataaaaaaggagacatgaacgatgattcaaaaacgaaagcggacag tttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagccgtagaccac tacatatgtgccaagaaaggggggacctgcaacttctccccctgcccgctcttcaacaggattgaagggacctgtta cagtggcaaggccaagtgctgcatccgctgaggatcc。

BamH Ⅰ

Further, in described step (4), bacillus subtilis uses B.subtilis ROTA9300 to be Host Strains.

Further, in described step (4), method of attachment uses the DNA Ligation Kit Ver2.1 method of TAKARA Operation is described, method for transformation illustrates operation according to the method for the E.coli DH5 α Competent Cells of TAKARA.

Further, in described step (5), LB culture medium is: tryptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, chloromycetin 10 μ g/mL, pH are nature.

Further, every a monoclonal positive transformant is added by described step (5) in the LB culture medium of 5mL.

Further, in described step (5), derivant is sucrose.

The beneficial effects of the present invention is: the invention provides a kind of porcine defensin and express on bacillus subtilis Method, the expression obtained reaches 3mg/L；The present invention is porcine defensin restructuring in bacillus subtilis and amalgamation and expression Providing theoretical foundation, establish the method that bacillus subtilis secretion expresses antibacterial peptide, the source solving antibacterial peptide limits Etc. problem.

Accompanying drawing explanation

Fig. 1 is the remodeling method schematic diagram of porcine defensin BD gene；

Fig. 2 is the structural representation of plasmid vector pHT43；

Fig. 3 is the synthetic route schematic diagram of recombinant expression carrier pHT-BD；

Fig. 4 is the positive colony PCR figure of recombinant expression carrier pHT-BD；

Fig. 5 is SDS-PAGE analytic process testing goal protein excretion band of expression figure；

The albumen antibacterial figure to staphylococcus aureus for the purpose of Fig. 6；

For the purpose of Fig. 7, albumen is to colibacillary antibacterial figure.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment to the present invention It is specifically described, it is necessary to it is noted that following example are used only for being explained and illustrated the present invention, be not used to Limit the present invention.Some nonessential improvement and adjustment that those skilled in the art are made according to foregoing invention content, still belong to In protection scope of the present invention.

Embodiment

It is prepared as follows porcine defensin fusion protein and expresses:

(1) transferring known porcine defensin BD gene order from ncbi database, its sequence is as follows:

gaccactacatatgtgccaagaaaggggggacctgcaacttctccccctgcccgctcttcaacaggattgaagggac ctgttacagtggcaaggccaagtgctgcatccgctga；

(2) synthesis of genes of interest

Choose above-mentioned porcine defensin BD gene to transform, BD gene adds SacB and SamyQ gene, remodeling method As it is shown in figure 1, obtain engineered protein SS-BD gene；5 ' the ends at engineered protein SS-BD gene add restricted enzyme KpnI Restriction enzyme site, the 3 ' ends of SS-BD add restricted enzyme BamH I restriction enzyme site, carry out double digestion reaction, it is thus achieved that SS-BD mesh Genetic fragment；Enzymatic cleavage methods uses TAKARA enzyme action system and explanation to operate, by Shanghai Invitrogen^TMCompany is straight It is bonded into；

Gained SS-BD genes of interest fragment sequence is as follows:

KpnI

BamH Ⅰ

(3) structure of recombinant expression carrier

Bacillus subtilis plasmid vector pHT43 is carried out KpnI/BamH I double digestion, obtains pHT43 double digestion gene Fragment, enzymatic cleavage methods uses TAKARA enzyme action system and explanation to operate；Then be attached with SS-BD genes of interest fragment and Convert, obtain recombinant expression carrier pHT-BD；Method of attachment uses the DNA Ligation Kit Ver2.1 method of TAKARA to say Bright operation, method for transformation illustrates operation according to the method for the E.coli DH5 α Competent Cells of TAKARA；Plasmid vector The schematic diagram of pHT43 as in figure 2 it is shown, the synthesis schematic diagram of recombinant expression carrier pHT-BD as shown in Figure 3.

Connection is converted the carrier pHT-BD obtained verify, by PCR positive colony acquired results as shown in Figure 4, Prove that pHT43 and SS-BD has connected to convert successfully.

(4) conversion of bacillus subtilis and screening

Taking recombinant expression carrier pHT-BD, the method provided by MoBiTec company is transformed into B.subtilis On ROTA9300, filter out the positive transformant being grown on chloromycetin flat board；

(5) cultivation and abduction delivering

It is inoculated in after the positive transformant filtered out is selected a monoclonal in the LB culture medium of 5mL, LB culture medium prescription For: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, chloromycetin 10 μ g/mL, pH are nature；It is subsequently placed in and shakes Bed, 37 DEG C, incubated overnight under the conditions of 180rpm, obtains one-level culture fluid, is inoculated in by the inoculum concentration of 1% by one-level culture fluid In 50mL above-mentioned LB culture medium, be placed in shaking table, 37 DEG C, cultivate under the conditions of 225rpm, to OD₆₀₀Reach to add derivant when 0.8 Sucrose to concentration is 2% (m/v), induced reaction 24h at 37 DEG C.

(6) expression of porcine defensin albumen is measured, takes the product 1mL after induced reaction, use SDS-PAGE Methods analyst identifies the secreting, expressing situation of destination protein, and the situation of testing goal band is as shown in Figure 5.

As seen in Figure 5, after being induced by derivant sucrose, have high-visible in destination protein size position Band, illustration purpose albumen by success secretion inducing express, after testing, the expression of destination protein is 3mg/L.

Test case

The bacteriostatic activity detection of destination protein:

After destination protein BD gene lyophilization is concentrated, blue respectively as leather with escherichia coli and staphylococcus aureus The bacteriostatic activity of indicator bacteria testing goal fragment BD of family name's negative bacterium and gram positive bacteria, method is to be existed by bacteria suspension Supernatant is collected, after supernatant concentration being become powder by vacuum lyophilization, with aseptic after centrifugal 10min under 12000rpm Powder is configured to sample liquid according to the concentration of 0.2g/mL by distilled water, then by Agar diffusion test Activity determination, wherein:

A group positive control: for the corresponding antibiotic of different indicator bacterias；

B group test sample: through the supernatant powder of the vacuum lyophilization of sucrose induction

C group negative control: without the supernatant powder of the vacuum lyophilization of sucrose induction

As shown in Figure 6 and Figure 7, wherein Fig. 6 is the testing result to gram positive bacteria to acquired results, and Fig. 7 is to leather orchid The testing result of family name's negative bacterium.

Can be seen that B group all possesses fungistatic effect to gram positive bacteria and negative bacterium by Fig. 6 and Fig. 7, C group is the most right Two kinds of indicator bacterias all do not have fungistatic effect.

Claims

1. the method utilizing bacillus subtilis height efficient expression porcine defensin, it is characterised in that comprise the steps:

(2) two ends of step (1) gained engineered protein SS-BD gene are carried out KpnI/BamH I double digestion and obtains SS-BD purpose Genetic fragment；

(3) select bacillus subtilis plasmid vector pHT43 to carry out KpnI/BamH I double digestion, obtain pHT43 double digestion carrier Genetic fragment；

(4) step (2) gains are attached with step (3) gains and convert, obtain recombinant vector pHT-BD, will restructuring Carrier pHT-BD is transformed on bacillus subtilis, the positive transformant that screening can be grown on chloromycetin flat board；

(5) it is inoculated in LB culture medium after the positive transformant monoclonal that will filter out, is placed in shaking table, at 37 DEG C, 180rpm bar Incubated overnight under part, obtains one-level culture fluid, by one-level culture fluid as 1% inoculum concentration be inoculated in LB culture medium described in 50mL In, be placed in shaking table, 37 DEG C, cultivate under the conditions of 225rpm, to OD₆₀₀Add derivant when reaching 0.8 to be calculated as to concentration by m/v 2%, induced reaction 24h at 37 DEG C.

Method the most according to claim 1, it is characterised in that the sequence of described porcine defensin BD gene is as follows:

gaccactacatatgtgccaagaaaggggggacctgcaacttctccccctgcccgctcttcaacaggattgaag ggacctgttacagtggcaaggccaagtgctgcatccgctga。

Method the most according to claim 1, it is characterised in that described step (2) is at the 5 ' of engineered protein SS-BD gene End adds KpnI restriction enzyme site, adds BamH I restriction enzyme site at 3 ' ends.

Method the most according to claim 3, it is characterised in that the sequence of described SS-BD genes of interest is as follows:

KpnI 。

ggtaccccttttatgattttctatcaaacaaaagaggaaaatagaccagttgcaatccaaacgagagtctaat agaatgaggtcgaaaagtaaatcgcgcgggtttgttactgataaagcaggcaagacctaaaatgtgtaaagggcaaa gtgtatactttggcgtcaccccttacatattttaggtctttttttattgtgcgtaactaacttgccatcttcaaaca ggagggctggaagaagcagaccgctaacacagtacataaaaaaggagacatgaacgatgattcaaaaacgaaagcgg acagtttcgttcagacttgtgcttatgtgcacgctgttatttgtcagtttgccgattacaaaaacatcagccgtaga ccactacatatgtgccaagaaaggggggacctgcaacttctccccctgcccgctcttcaacaggattgaagggacct gttacagtggcaaggccaagtgctgcatccgctgaggatcc。

BamH Ⅰ。

Method the most according to claim 1, it is characterised in that in described step (4), bacillus subtilis uses B.subtilis ROTA9300 is Host Strains.

Method the most according to claim 1, it is characterised in that in described step (4), method of attachment uses the DNA of TAKARA The explanation operation of Ligation Kit Ver2.1 method, method for transformation is according to the E.coli DH5 α Competent of TAKARA The method explanation operation of Cells.

Method the most according to claim 1, it is characterised in that in described step (5), LB culture medium is: tryptone 10g/ L, yeast extract 5g/L, sodium chloride 10g/L, chloromycetin 10 μ g/mL, pH are nature.

Method the most according to claim 1, it is characterised in that will every a monoclonal positive transformants in described step (5) Son adds in the LB culture medium of 5mL.

Method the most according to claim 1, it is characterised in that in described step (5), derivant is sucrose.