CN104131022A - Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof - Google Patents

Clostridium perfringens alpha, beta 1, beta 2 and epsilon coexpression vector and construction method and expression method thereof Download PDF

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CN104131022A
CN104131022A CN201410378016.6A CN201410378016A CN104131022A CN 104131022 A CN104131022 A CN 104131022A CN 201410378016 A CN201410378016 A CN 201410378016A CN 104131022 A CN104131022 A CN 104131022A
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toxin
carrier
encoding gene
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周继章
韩广伟
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Lanzhou Veterinary Research Institute of CAAS
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Abstract

The invention discloses a clostridium perfringens alpha, beta 1, beta 2 and epsilon toxin coexpression vector; vectors are respectively pETDuet-1 and pRSFDuet-1 expressed in the same host bacteria, and the vectors are respectively connected with clostridium perfringens alpha and epsilon toxin encoding genes and clostridium perfringens beta 1 and beta 2 toxin encoding genes. On the basis, the invention also discloses a construction method and an expression method of the coexpression vector.

Description

Perfringens alpha, β 1, β 2, ε toxin co-expression carrier and construction process and expression method
Technical field
The present invention relates to a kind of co-expression carrier and structure and expression method of utilizing many toxin that gene engineering obtains, belong to bioengineering field.
Background technology
The zoonotic prevention and control of many serotype are one of major reasons of puzzle veterinary science worker, find and can express multiple protein simultaneously, and the immunogenicity of albumen is not damaged and changes, the subunit vaccine that simultaneously various serotype is played to prevention and control effect is that the technical problem solving is badly in need of in current this area.
Clostridium perfringens (clostridium perfringens), Gram-positive is produced gemma anaerobic bacillus(cillus anaerobicus), be distributed widely in occurring in nature, all visible in soil, sewage, food, ight soil etc., it is also the normal integral part of human intestine's flora simultaneously.The pathogenic effects of this bacterium is mainly reflected in the toxin aspect that it produces, and α, β, ε, ι are four kinds of main pathogenic toxin that this bacterium produces, and this bacterium can be divided on this basis to A, B, C, D, E5 type.Clostridium perfringens is a kind of important infecting both domestic animals and human disease pathogen, can cause the mankind's gas gangrene.Can cause in addition the many animals diseases such as sheep braxy, lamb dysentery, sheep enterotoxemia, piglet dysentery, calf enterotoxemia, livestock industry has been caused to huge financial loss.
The immunity of such disease now mainly realizes by the toxin producing in clostridium perfringens culturing process is processed into toxoid; Chinese invention patent application 200680021810.9 has been invented C. perfringens alpha toxoid vaccine, and proves that this vaccine can produce enough protection antibodies by induced animal.Although toxoid vaccine energy induced animal produces protection antibody; but the shortcomings such as security is not high, poor stability that it exists; these shortcomings have limited its application greatly; recombinant vaccine thereby become goal in research; recombinant vaccine only contains the necessary immunizing composition of generation protective immune response; containing the composition do not wanted of immunity, thereby security, good stability, also reduce in addition and eliminated the reaction such as pyrogen, allergen that conventional vaccine is difficult to avoid former.
The recombinant plasmid that Chinese invention patent application 200310123697.3 is utilized round pcr to build to contain β 1, β 2 fusion genes, and carry out the abduction delivering of fusion rotein, provide desirable genetic material for further developing multivalent genetic engineering subunit seedling, for the animal necrotic enteritis and the enterotoxemia that solve the livestock industry of puzzlement China provide a new approach.But this recombinant plasmid is the series connection of multiple toxin genes, can express at most three toxin simultaneously, in the time of abduction delivering, tend to occur expression amount deficiency, cause immune animal cannot produce enough immune protective efficiencies.Thereby, be badly in need of building the recombinant plasmid that can simultaneously express multiple toxin, for the preparation of clostridium polyvalent vaccine provides a kind of effectively approach.
Summary of the invention
For the deficiencies in the prior art, the invention discloses a kind of perfringens alpha, β 1, β 2, ε toxin co-expression carrier, utilize the means of molecular cloning by α, β 1, β 2, tetra-kinds of toxin of ε encoding gene be connected with dual-expression vector pETDuet-1, pRSFDuet-1 respectively, built co-expression carrier and can in same bacterial strain, express perfringens alpha, β simultaneously 1, β 2, tetra-toxin proteins of ε, can be used as multivalence clostridium perfringens genetic engineering subunit vaccine and be used for the zoonosis of the many serotypes of prevention and control, effectively to improve the Prevention Technique level of clostridial disease in China's farming animals, ensure that aquaculture develops in a healthy way, provides peasants and herdsmen to take in, ensure public health and livestock product safety.
For achieving the above object, the present invention is achieved through the following technical solutions:
Perfringens alpha, β 1, β 2, ε toxin co-expression carrier, carrier is respectively the pETDuet-1, the pRSFDuet-1 that in same Host Strains, express, is connected with respectively perfringens alpha, ε toxin encoding gene and clostridium perfringens β on carrier 1, β 2toxin encoding gene.
The present invention carrier pETDuet-1, pRSFDuet-1 used is dual-expression vector, on each carrier, there are two promotors, belong to single carrier double-promoter system, each toxin gene has an independently promotor, the expression of each each toxin protein of promotor independent drive.
Compared with traditional gene fusion construct, dual-expression vector of the present invention has been avoided influencing each other between gene, has ensured the expression amount that toxin protein is stable, has overcome the shortcoming of tandem expression expression amount deficiency.And by the double-plasmid expression system of expressing in same Host Strains, pETDuet-1 with pRSFDuet-1 as identical inconsistent two plasmids of replicon, under the pressure of Amp and two kinds of microbiotic existence of Kana, filial generation bacterium can effectively survive, can be by antibiotic kills, enter effectively expressing exotic toxin gene after same Host Strains thereby can transform simultaneously.
For the ease of large-scale production, reduce and cultivate difficulty, preferred the present invention's Host Strains used is intestinal bacteria, it cultivates simple, can adopt fermentor tank to accomplish scale production, thereby effectively control quality, ensures security and the validity of the vaccine of producing.
In conventional biotechnology, intestinal bacteria used all can be used for the present invention, include but not limited to e. coli bl21, BL21 (DE3), and BL21 (DE3) PLySs etc.
Present inventor has fully studied complexity and the accuracy of annexation between the encoding gene of different toxin proteins and expression vector in R&D process, and preferred annexation is pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2, α, ε toxin encoding gene insert dual-expression vector pETDuet-1, β 1, β 2toxin encoding gene inserts dual-expression vector pRSFDuet-1.
Accordingly, the invention discloses the construction process of above-mentioned co-expression carrier, by respectively by perfringens alpha and ε toxin encoding gene, clostridium perfringens β 1and β 2toxin encoding gene is connected with dual-expression vector pETDuet-1, pRSFDuet-1, builds co-expression carrier, and concrete comprises the steps:
1) utilize the DNA of clostridium perfringens bacterial strain to amplify α, β as PCR masterplate 1, β 2, ε toxin encoding gene;
2) use EcoRI, PstI double digestion two carrier pETDuet-1, pRSFDuet-1 respectively;
3) utilize EcoRI, PstI double digestion α, β respectively 2toxin gene is connected it to obtain expression vector with pETDuet-1, pRSFDuet-1 carrier after double digestion respectively under the effect of T4DNA ligase enzyme;
4) utilize BglII, XhoI double digestion step 3) expression vector that obtains, use BglII, XhoI double digestion ε, β 1toxin encoding gene is connected and obtains co-expression carrier with the expression vector after double digestion respectively under the effect of T4DNA ligase enzyme.
Wherein, step 1) α, β 1, β 2, the design of ε toxin encoding gene amplification the primer is as follows:
The primer of clostridium perfringens alpha toxin encoding gene is CPA-F:CTCGAATTCATGGGATGGAAAGATTGATG, CPA-R:GGCCTGCAGCCACCAAAACCAATATT;
Clostridium perfringens β 1the primer of toxin encoding gene is CPB1-F:CGCAGATCTCAATGATATAGGTAAAAC, CPB1-R:CCGCTCGAGATTAATAGCTGTTACTTTGTG;
Clostridium perfringens β 2the primer of toxin encoding gene is CPB2-F:CATGAATTCAGTTAAAGCAAATGAAGTGA, CPB2-R:CGCCTGCAGTAATAACAATAACCCTCACC;
The primer of clostridium perfringens ε toxin encoding gene is ETX-F:CGCAGATCTCAAGGAAATATCTAATACAGTA, ETX-R:CCGCTCGAGTTCTAATAAATAATCTTATTTT.
Above-mentioned pcr amplification reaction adopts conventional PCR reaction conditions, without particular requirement.
Above-mentioned four kinds of toxin protein coding gene sequences all can openly be found from database, also genome that can clostridium perfringens reference culture is that template amplification obtains, for example, can amplify α, β taking the genome of clostridium perfringens C59-1 as template by above-mentioned primer 1, β 2the encoding gene of toxin, can amplify the encoding gene of ε toxin by above-mentioned primer taking clostridium perfringens C60-2 genome as template.
As mentioned above, the annexation of preferred carrier and toxin encoding gene is step 3) build respectively pETDuet-1-α and pRSFDuet-1-β 2carrier, step 4) build respectively pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2carrier.
Construction process of the present invention, the encoding gene of α, ε toxin is inserted dual-expression vector pETDuet-1, β by the process of being cut, connect, being transformed by enzyme 1, β 2toxin encoding gene inserts dual-expression vector pRSFDuet-1, thereby can be used in efficient express alpha, β in same Host Strains 1, β 2, tetra-kinds of toxin proteins of ε, these four kinds of toxin proteins can with same animal body in positive serum react, there is good immunogenicity.
On the basis of the above, the invention also discloses described perfringens alpha, β 1, β 2, ε toxin co-expression carrier expression method, be by above-mentioned perfringens alpha, β 1, β 2, ε toxin co-expression carrier is transformed into Host Strains, picking mono-clonal colony inoculation, in LB substratum, shakes bacterium and spends the night, and is transferred in new LB substratum, adopts IPTG abduction delivering.
As aforementioned, Host Strains used is preferably intestinal bacteria, includes but not limited to e. coli bl21, BL21 (DE3), and BL21 (DE3) PLySs etc.
Above-mentioned expression method, by two carriers that carry four toxin genes are transformed and enter same Host Strains, can be stablized, efficient express alpha, β 1, β 2, tetra-kinds of toxin proteins of ε bacterial strain, its four kinds of expressed toxin proteins exist with inclusion body form, do not have toxin itself active, and have good immunogenicity and can produce good immune protective efficiency by induced animal body, resist 1 × LD 100the attack of clostridium perfringens.
Owing to thering is above-mentioned characteristic, the invention also discloses a kind of multivalence clostridium perfringens genetic engineering subunit vaccine, comprise and contain co-expression carrier pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2host Strains, because this vaccine antigen used is protein, can not occur to occur gene recombination problem compared with other vaccines (as live vector vaccine, nucleic acid vaccine, gene-deleted vaccine), therefore not have biological safety problem, be applicable to biological use in enormous quantities.
Brief description of the drawings
Fig. 1 is α, β 1, β 2, ε toxin gene amplification electrophorogram, wherein M is 2000bp Marker, 1-4 is respectively α, ε, β 1, β 2toxin encoding gene;
Fig. 2 has shown α, ε, β 1, β 2toxin gene double digestion qualification figure, wherein, A is α, ε toxin gene double digestion qualification figure, wherein M is 2000bp Marker, the 1st, co-expression carrier pETDuet-1-α-ε process EcoRI and PstI double digestion result, the 2nd, co-expression carrier pETDuet-1-α-ε is through BglII and XhoI double digestion result; B is β 1, β 2toxin gene double digestion qualification figure, wherein M is 2000bp Marker, the 1st, co-expression carrier pRSFDuet-1-β 1-β 2through EcoRI and PstI double digestion result, the 2nd, co-expression carrier pRSFDuet-1-β 1-β 2through BglII and XhoI double digestion result;
Fig. 3 is α, ε, β 1, β 2the SDS-PAGE figure that four toxin proteins are expressed, wherein M is albumen Marker, the 1st, proceed to the full cell lysate of BL21 (DE3) of empty plasmid pETDuet-1 and pRSFDuet-1 simultaneously; The 2nd, proceed to pETDuet-1-α-ε and pRSFDuet-1-β simultaneously 1-β 2co-expression carrier does not add the full cell lysate of BL21 (DE3) of IPTG induction; The 3rd, co-expression carrier pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2proceed in BL21 (DE3) through four toxin proteins of 0.8mM IPTG abduction delivering the figure of the SDS-PAGE after 4 hours simultaneously.
Fig. 4 is α, ε, β 1, β 2the immunoblotting qualification of four toxin, wherein A-2 uses α, the ε toxin Western blotting qualification of D type clostridium perfringens positive serum to coexpression, and B-2 uses α, ε, the β of Type B clostridium perfringens positive serum to coexpression 1, β 2the Western blotting qualification of toxin.
Embodiment
The main raw that used in the present invention all can be bought from associated biomolecule goods company, has provided the purchase source of middle material therefor in implementing below, is only signal.
Clostridium perfringens bacterial strain: C59-1, C60-2 are purchased from China Veterinery Drug Inspection Office;
PETDuet-1 and pRSFDuet-1 dual-expression vector are purchased from Novagen company;
Express bacterium:
BL21 (DE3) is the expressive host bacterium of four toxin proteins, purchased from Beijing Quanshijin Biotechnology Co., Ltd.
The primer is synthetic by Shanghai Sheng Gong biotechnology company limited;
Enzyme used and reagent: restriction enzyme BglII, EcoRI, PstI, XhoI, T4DNA ligase enzyme and 2 × Power Taq PCR MasterMix are all purchased from the precious biological company limited in Dalian; IPTG, SDS (sodium laurylsulfonate), 2000DNA Marker, Agarose DNA extraction kit, DNA fast purifying reclaim test kit, plasmid rapid extraction test kit all purchased from Dalian Bao Bio-Engineering Company; It is synthetic purchased from Shanghai Sheng Gong biotechnology company limited that bacterial genomes is extracted test kit; Agarose is purchased from Invitrogen company; Dye in advance albumen Marker purchased from Fermentas company.
Embodiment 1: perfringens alpha, β 1, β 2, ε toxin co-expression carrier structure and expression
1. α, ε, β 1, β 2the amplification of toxin encoding gene
By buying the clostridium perfringens reference culture C59-1 of preservation freeze-drying that comes and C60-2 and inoculate incubated overnight in the lonely liver soup substratum of fresh configuration, work as OD 600the lonely liver soup nutrient solution of aseptic collection while reaching 0.6-0.8, utilizes bacterial genomes to extract test kit extraction C59-1 and C60-2 genomic dna amplifies each toxin gene as PCR masterplate.
PCR reaction system: 50 μ L MasterMix, the each 2 μ L of upstream and downstream primer, masterplate 16 μ L, deionized water 30 μ L; DNA glue reclaims test kit and reclaims four toxin encoding genes;
2.pETDuet-1-α and pRSFDuet-1-β 2the structure of carrier
PETDuet-1, two carriers of pRSFDuet-1 are carried out to EcoRI, PstI double digestion, 40 μ L enzymes are cut system: 10 × H damping fluid, 4 μ L, the each 1 μ L of EcoRI, PstI, carrier 24 μ L, deionized water 8 μ L, after enzyme is cut product 1% agarose electrophoresis, glue reclaims test kit recovery;
α, the β of EcoRI, PstI double digestion 2toxin gene, 40 μ L enzymes are cut system: 10 × H damping fluid, 4 μ L, the each 1 μ L of EcoRI, PstI, goal gene 24 μ L, deionized water 8 μ L, after enzyme is cut product 1% agarose electrophoresis, glue reclaims test kit recovery, then be connected with the pETDuet-1 of double digestion, the carrier of pRSFDuet-1 respectively, build two linked systems: 10 × T4DNA Ligase Buffer2.5 μ L,, alpha toxin encoding gene glue reclaims product 14 μ L, pETDuet-12 μ L, T4DNA Ligase1 μ L, deionized water 5.5 μ L; 10 × T4DNA Ligase Buffer2.5 μ L,, β 2toxin encoding gene glue reclaims product 14 μ L, pRSFDuet-1 2 μ L, T4DNA Ligase1 μ L, deionized water 5.5 μ L.16 DEG C of connections are spent the night, and transform respectively Trans109 state cell, and picking mono-clonal bacterium colony carries out respectively bacterium liquid PCR qualification, enzyme is cut qualification, the order-checking of bacterium liquid definite α, β 2be connected respectively to pETDuet-1-α and pRSFDuet-1-β 2on carrier;
3.pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2the structure of carrier
PETDuet-1-α and pRSFDuet-1-β 2carry out BglII, XhoI double digestion, 40 μ L enzymes are cut system: 10 × H damping fluid, 4 μ L, and the each 1 μ L of EcoRI, PstI, carrier 24 μ L, deionized water 8 μ L, after enzyme is cut product 1% agarose electrophoresis, glue reclaims test kit recovery.
ε, the β of BglII, XhoI double digestion 1toxin encoding gene, 40 μ L enzymes are cut system: 10 × H damping fluid, 4 μ L, the each 1 μ L of EcoRI, PstI, goal gene 24 μ L, deionized water 8 μ L, after enzyme is cut product 1% agarose electrophoresis, glue reclaims test kit recovery, then builds two link systems: 10 × T4DNA Ligase Buffer2.5 μ L, ε toxin encoding gene glue reclaims product 14 μ L, pETDuet-1-α 2 μ L, T4DNA Ligase1 μ L, deionized water 5.5 μ L; 10 × T4DNA Ligase Buffer2.5 μ L,, β 1toxin encoding gene glue reclaims product 14 μ L, pRSFDuet-1-β 22 μ L, T4DNA Ligase1 μ L, deionized water 5.5 μ L.16 DEG C of connections are spent the night, and transform respectively Trans109 state cell, and picking mono-clonal bacterium colony carries out respectively bacterium liquid PCR qualification, enzyme is cut qualification, the order-checking of bacterium liquid definite ε, β 1be connected respectively to pETDuet-1-α and pRSFDuet-1-β 2on carrier;
4. α, ε, β 1, β 2the coexpression of four toxin
PETDuet-1-α and pRSFDuet-1-β 2each 0.5 μ L plasmid transforms BL21 (DE3) Host Strains simultaneously, and picking mono-clonal colony inoculation, in fresh 10ml LB substratum, shakes bacterium and spends the night, transfer in fresh 10mlLB substratum in 1: 100 ratio, and OD 600when value reaches 0.6-0.8, get 1ml bacterium liquid as not inducing sample, then add the 10 μ L IPTG abduction deliverings of final concentration 0.8mM within four hours, to collect 1ml bacterium liquid sample.
Embodiment 2: α, ε, β 1, β 2the coexpression result of four toxin detects
The preparation of 1 SDS-PAGE solution:
Tris-glycine buffer: 25mmol/L Tris, 250mmol/L glycine (pH8.0), 0.1%SDS.
2 × SDS loading buffer:100mmol/L Tris.C1 (pH8.0), 200m mol/L mercaptoethanol, 4%SDS, 0.2% tetrabromophenol sulfonphthalein, 20% glycerine.
Coomassie brilliant blue staining liquid: dissolve 0.25g coomassie brilliant blue R250 in 45ml methyl alcohol, 45ml water, 10ml Glacial acetic acid.
Destainer: 30% methyl alcohol, 10% glacial acetic acid, distilled water complement amasss to 100ml.
2 SDS-PAGE experiments
(1) clean sheet glass, be fixed on electrophoresis chamber, with 2.0% agarose edge sealing.Press the separation gel 5ml of the formulated 15% of molecular cloning, inject rapidly between two sheet glass, Jiao Ding covers 1ml distilled water.The solid hypsokinesis of gelling to be separated goes out tectum liquid, with deionized water rinsing gel top several, is inverted empty dry liquids, adds the concentrated sol solution of 2ml 5%, plugs comb, vertically places electrophoresis chamber it is solidified.
(2) carefully remove comb, between the upper and lower groove of electrophoresis apparatus, add Tris-glycine buffer, with irrigation with syringe well several, power cathode is connected with upper groove.
(3) the centrifugal 2min of bacterium liquid 12000rpm collecting, with the PBS suspension of 50 μ l pH7.4, add isopyknic 2 × SDS loading buffer, mix, 10min is boiled in water-bath, with microsyringe loading 10 μ l, opening power, enter separation gel by 80V voltage electrophoresis to tetrabromophenol sulfonphthalein, voltage is brought up to 120V, continue electrophoresis to tetrabromophenol sulfonphthalein and arrive gel bottom.
(4) dyeing: take off gel distilled water flushing, with the coomassie brilliant blue staining immersion foaming gel of 5 times of gel volumes, be placed on room temperature dyeing 3h on decolorization swinging table.
(5) decolouring: with the mixed solution of 30% methyl alcohol, 10% glacial acetic acid, decolour and spend the night on decolorization swinging table, change staining fluid therebetween 3～4 times.After blue background purifies completely, gel is immersed in distilled water and stops decolouring.

Claims (9)

1. perfringens alpha, β 1, β 2, ε toxin co-expression carrier, it is characterized in that carrier is respectively pETDuet-1, the pRSFDuet-1 expressing in same Host Strains, is connected with respectively perfringens alpha, ε toxin encoding gene and clostridium perfringens β on carrier 1, β 2toxin encoding gene.
2. co-expression carrier according to claim 1, is characterized in that described Host Strains is intestinal bacteria.
3. co-expression carrier according to claim 1, is characterized in that annexation is pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2.
4. perfringens alpha, β 1, β 2, ε toxin co-expression carrier construction process, it is characterized in that respectively perfringens alpha and ε toxin encoding gene, clostridium perfringens β 1and β 2toxin encoding gene is connected with dual-expression vector pETDuet-1, pRSFDuet-1, builds co-expression carrier.
5. construction process according to claim 4, is characterized in that comprising the steps: 1) utilize the DNA of clostridium perfringens bacterial strain to amplify α, β as PCR masterplate 1, β 2, ε toxin encoding gene; 2) use EcoRI, PstI double digestion two carrier pETDuet-1, pRSFDuet-1 respectively; 3) utilize EcoRI, PstI double digestion α, β respectively 2toxin gene is connected it to obtain expression vector with pETDuet-1, pRSFDuet-1 carrier after double digestion respectively under the effect of T4DNA ligase enzyme; 4) utilize BglII, XhoI double digestion step 3) expression vector that obtains, use BglII, XhoI double digestion ε, β 1toxin encoding gene is connected and obtains co-expression carrier with the expression vector after double digestion respectively under the effect of T4DNA ligase enzyme.
6. construction process according to claim 5, is characterized in that step 1) amplification the primer be respectively:
The primer of clostridium perfringens alpha toxin encoding gene is CPA-F:CTCGAATTCATGGGATGGAAAGATTGATG, CPA-R:GGCCTGCAGCCACCAAAACCAATATT;
Clostridium perfringens β 1the primer of toxin encoding gene is CPB1-F:CGCAGATCTCAATGATATAGGTAAAAC, CPB1-R:CCGCTCGAGATTAATAGCTGTTACTTTGTG;
Clostridium perfringens β 2the primer of toxin encoding gene is CPB2-F:CATGAATTCAGTTAAAGCAAATGAAGTGA, CPB2-R:CGCCTGCAGTAATAACAATAACCCTCACC;
The primer of clostridium perfringens ε toxin encoding gene is ETX-F:CGCAGATCTCAAGGAAATATCTAATACAGTA, ETX-R:CCGCTCGAGTTCTAATAAATAATCTTATTTT.
7. construction process according to claim 5, the annexation that it is characterized in that carrier and toxin encoding gene is step 3) build respectively pETDuet-1-α and pRSFDuet-1-β 2carrier, step 4) build respectively pETDuet-1-α-ε and pRSFDuet-1-β 1-β 2carrier.
8. perfringens alpha, β 1, β 2, ε toxin co-expression carrier expression method, it is characterized in that the perfringens alpha of claim 1, β 1, β 2, ε toxin co-expression carrier is transformed into Host Strains, picking mono-clonal colony inoculation, in LB substratum, shakes bacterium and spends the night, and is transferred in new LB substratum, adopts IPTG abduction delivering.
9. expression method according to claim 8, is characterized in that described Host Strains is intestinal bacteria.
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CN106987548A (en) * 2017-04-10 2017-07-28 东北农业大学 It is a kind of to express perfringens alpha, β 2, the ectotoxic recombinant Lactobacillus of ε, β 1 and its construction method and application simultaneously
CN106987548B (en) * 2017-04-10 2020-12-18 东北农业大学 Recombinant lactobacillus for simultaneously expressing alpha, beta 2 and beta 1 exotoxins of clostridium perfringens and construction method and application thereof
CN107012239A (en) * 2017-05-06 2017-08-04 雷宇 A kind of multiplex PCR classifying method of C.perfringens
CN111808202A (en) * 2020-09-11 2020-10-23 苏州世诺生物技术有限公司 Clostridium perfringens gene engineering subunit vaccine, preparation method and application thereof
CN113702640A (en) * 2021-06-16 2021-11-26 宁夏大学 Indirect ELISA method for clostridium perfringens beta 1 toxin antibody

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