CN103861094A - Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof - Google Patents
Clostridium perfringen alpha toxin genetic engineering vaccine and application thereof Download PDFInfo
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Abstract
The invention relates to a clostridium perfringen alpha toxin genetic engineering vaccine and application thereof. The clostridium perfringen alpha toxin genetic engineering vaccine is prepared by designing a specific primer, implementing polymerase chain reaction (PCR) amplification on A-type clostridium perfringen DNA to obtain an alpha toxin gene segment, cloning the gene segment onto a pMD18-T Vector, selecting a positive recon and linking the positive recon onto a PET-28 alpha (+) plasmid; introducing the recombinant plasmid to a receptor strain to successfully construct a recombinant strain BL21 (DE3)-alpha; emulsifying the prepared alpha toxin protein and an aluminum hydroxide adjuvant at a volume ratio of 9:1, and adding 0.01% thiomersal to obtain the clostridium perfringen alpha toxin genetic engineering vaccine. The clostridium perfringen alpha toxin genetic engineering vaccine is easy for realization of industrial production, simple to operate and good in safety, and is capable of effectively preventing such diseases as animal necrotic enteritis, enterotoxemia, and human and animal traumatic gas gangrene caused by the A-type clostridium perfringens.
Description
(1) technical field
A kind of clostridium perfringens alpha toxin recombinant vaccine of the present invention and application thereof, belong to animal gene engineering technology field.
(2) background of invention
Bacillus perfringens is to cause one of the main pathogenic fungi that various animal necrotic enteritis, enterotoxemia, human and animal trauma gas gangrene and human foods are poisoning, and the virulence factor of this bacterium is its extracellular toxin producing.So far, the bacillus perfringens extracellular toxin of having found has more than 12 kinds (α, β, γ, δ, ε, η, θ, ι, κ, λ, μ and ν), but only have 4 kinds (α, β, ε and the ι) that play main pathogenic effects, are divided into this bacterium 5 toxin types such as A, B, C, D, E accordingly.Various bacillus perfringens all produces alpha toxin, it is the main virulence factor of bacillus perfringens, there is the activity of 2 kinds of enzymes such as phospholipase C and sphingomyelinase, can be hydrolyzed phosphatidylcholine and sphingomyelins on cell membrane simultaneously, cause lysis, thereby there is the characteristics such as cytotoxicity, hemolytic activity, lethal and platelet aggregation, cause animal gas gangrene, rabbit clostridium property dysentery etc., the health of serious threat poultry, hinders the development of animal husbandry.
In order to control the popular of bacillus perfringens disease, many scientific research scholars once developed the inactivated vaccine of bacillus perfringens for prevention.
wang Pu(2013) preparation and the Humoral evaluation study with the concealed bacillus bivalent inactivated vaccine that suppurates to bacillus perfringens;
guo Fenghuadevelopment research Deng (2010) to A type bacillus perfringens toxoid inactivated vaccine;
qu Haibodeng (2010) to A type bacillus perfringens toxoid vaccine and the full safety of bacterium inactivated vaccine and the comparative study of immune efficacy; Xu is the development of medium (2005) rabbit hemorrhagic disease, pasteurella multocida disease, the sick A type of bacillus perfringens triple inactivated vaccine.These vaccines have certain control action to the popular of this disease, but it exists following shortcoming: the immunoreation persistent period that induction produces is shorter, need to repeatedly inoculate; Inactivator contratoxin antigen has impact, and different on different antigenic component impacts; Because the immunoreation level of induction is lower, and vaccine between each antigenic component replys imbalance, may induce an illness; General Requirements carries out concentrated and purified to vaccine; Can not inoculate by natural way, be difficult for producing local immunity reaction etc., quality requires further improvement.
The toxoid vaccine of alpha toxin has protective effect on animal; but be unlikely applicable to the mankind for this vaccine of security consideration; therefore the understanding completely that vaccine research must be based on contratoxin structure and molecular function, payes attention to the interaction relationship on toxin and respiratory tract and gastrointestinal tract surface.The promoter of alpha toxin is one of the strongest promoter, can be identified by colibacillary RNA polymerase, thereby alpha toxin not only can be in bacillus perfringens itself under self promoter effect high efficient expression, also can in escherichia coli, obtain high efficient expression.The present invention is according to the pathogeny of bacillus perfringens; obtain the protective antigen gene fragment of alpha toxin; utilize Protocols in Molecular Biology; develop alpha toxin recombinant vaccine; this vaccine output is high, safe, purity is high, good stability, can effectively avoid the problems such as vaccine is concentrated, the induction of immunity persistent period is short.
(3) summary of the invention
The object of the present invention is to provide a kind of clostridium perfringens alpha toxin recombinant vaccine and application thereof, this vaccine possesses the advantages such as immunity is strong, specific aim good, have no side effect, safety is good, can effectively prevent the diseases such as the caused wounds in animals gas gangrene of A type bacillus perfringens, malignant edema, necrotic enteritis, enterotoxemia.
A kind of clostridium perfringens alpha toxin recombinant vaccine, is that the ratio take volume ratio as 9:1 is carried out emulsifying by clostridium perfringens alpha toxin albumen and aluminum hydroxide adjuvant, adds 0.01 gram of thimerosal to obtain in every 100ml emulsion.
The preparation process of described clostridium perfringens alpha toxin albumen is as follows:
1) design primer
According to the alpha toxin gene order (number of logging in: AY823400 of having submitted on Genebank, its sequence is as shown in Seq ID No:1) amplification alpha toxin part specific gene fragment, design primer: primer1 and primer2, and add respectively Nco I and BamH I restriction enzyme site;
primer1：5'-AGGGATGGAAAGATTGATGG-3' Nco I
primer2：5'-TTATTTTATATTATAAGTTGAATTTCCTGA-3' BamH I
2) prepare template and pcr amplification genes of interest
With inoculating loop picking A type bacillus perfringens strain, be inoculated in a blood plate culture medium, 37 ℃ of anaerobism are cultivated 36h; Get single colony inoculation and contain 7-8 milliliter THIOGLYCOLLIC ACID salt broth in vitro, 37 ℃ of anaerobism cultivation 12h, cultivation bacterium liquid extraction DNA profiling in one; Pcr amplification genes of interest, amplified production, after agarose gel electrophoresis, reclaims amplified production;
3) alpha toxin target DNA is cloned into pMD18-T Vector carrier
By step 2) in the extension amplification outcome that reclaims to pMD18-T Vector, after clone products is mixed with Top10 competent cell, transform, screen recombinant conversion bacterium extraction plasmid pMD18-T-α; Determine positive recombiant plasmid pMD18-T-α through enzyme action order-checking;
4) build prokaryotic expression carrier pET28a-α
By positive recombiant plasmid pMD18-T-α and expression vector pet28a double digestion, enzyme action product, through 1% agarose gel electrophoresis, reclaims object band and pet28a linear carrier; To reclaim product and be connected with pet28a linear carrier, build prokaryotic expression carrier pET28a-α; After pET28a-α is mixed with BL21 (DE3) pLysS competent cell, transform, obtain recombinant bacterial strain BL21 (DE3)-α;
5) abduction delivering of alpha toxin albumen, purification
With the IPTG of 0.1mM, 37 ℃, to recombinant bacterium abduction delivering 12h, the purified alpha toxin albumen that obtains.
The preparation method that the invention still further relates to a kind of clostridium perfringens alpha toxin recombinant vaccine, comprises the steps:
1) design primer
According to the alpha toxin gene order (number of logging in: AY823400 of having submitted on Genebank, its sequence is as shown in Seq ID No:1) amplification alpha toxin part specific gene fragment, design primer: primer1 and primer2, and add respectively Nco I and BamH I restriction enzyme site;
primer1：5'-AGGGATGGAAAGATTGATGG-3' Nco I
primer2：5'-TTATTTTATATTATAAGTTGAATTTCCTGA-3' BamH I
2) prepare template and pcr amplification genes of interest
With inoculating loop picking A type bacillus perfringens strain, be inoculated in a blood plate culture medium, 37 ℃ of anaerobism are cultivated 36h; Get single colony inoculation and contain 7-8 milliliter THIOGLYCOLLIC ACID salt broth in vitro, 37 ℃ of anaerobism cultivation 12h, cultivation bacterium liquid extraction DNA profiling in one; Pcr amplification genes of interest, amplified production, after agarose gel electrophoresis, reclaims amplified production;
3) alpha toxin target DNA is cloned into pMD18-T Vector carrier
By step 2) in the extension amplification outcome that reclaims to pMD18-T Vector, after clone products is mixed with Top10 competent cell, transform, screen recombinant conversion bacterium extraction plasmid pMD18-T-α; Determine positive recombiant plasmid pMD18-T-α through enzyme action order-checking;
4) build prokaryotic expression carrier pET28a-α
By positive recombiant plasmid pMD18-T-α and expression vector pet28a double digestion, enzyme action product, through 1% agarose gel electrophoresis, reclaims object band and pet28a linear carrier; To reclaim product and be connected with pet28a linear carrier, build prokaryotic expression carrier pET28a-α; After pET28a-α is mixed with BL21 (DE3) pLysS competent cell, transform, obtain recombinant bacterial strain BL21 (DE3)-α;
5) abduction delivering of alpha toxin albumen, purification
With the IPTG of 0.1mM, 37 ℃, to recombinant bacterium abduction delivering 12h, the purified alpha toxin albumen that obtains.
6) by described alpha toxin albumen and aluminum hydroxide adjuvant, the ratio take volume ratio as 9:1 is carried out emulsifying, adds 0.01 gram of thimerosal to obtain clostridium perfringens alpha toxin recombinant vaccine in every 100ml emulsion.
The invention still further relates to clostridium perfringens alpha toxin recombinant vaccine in prevention because of the application in the wounds in animals gas gangrene of the caused animal of A type bacillus perfringens, malignant edema, necrotic enteritis, enterotoxemia.
The outstanding behaviours of above-mentioned immune effect of clostridium perfringens alpha toxin recombinant vaccine is in the application of chicken and rabbit; When for rabbit immunity, immunizing dose is 2ml/, intramuscular injection; When for chicken immune, immunizing dose is 0.5ml/, cervical region subcutaneous injection.
Compared with prior art, the present invention has following beneficial effect:
Clostridium perfringens alpha toxin recombinant vaccine disclosed by the invention, possess that immunity is strong, specific aim good, have no side effect, the advantage such as safety is good, purity is high, good stability and can avoid the problems such as vaccine is concentrated, the induction of immunity persistent period is short, effectively prevent the diseases such as the caused wounds in animals gas gangrene of A type bacillus perfringens, malignant edema, necrotic enteritis, enterotoxemia.
(4) accompanying drawing explanation
Fig. 1 is the pcr amplification result figure of the object of the invention gene, illustrates that PCR successfully amplifies the alpha toxin gene of 1113bp.
Fig. 2 is protein SDS-PAGE electrophoresis result figure after Ni-NTA post of the present invention is purified, the bacterium liquid ultrasonic degradation of inducing under optimal conditions is described, supernatant and deposit sample be purification respectively, and after purification, protein sample is through SDS-PAGE electrophoresis, target protein mainly exists with the form of inclusion body, big or small about 45KD.
Fig. 3 is the Western blot testing result figure of alpha toxin expressing protein of the present invention, and result shows that destination protein can react with positive antibody, illustrates that the alpha toxin albumen of expressing has good specificity.
(5) specific embodiment
Further explain the present invention below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.The all culture medium that relate in an embodiment and molecular biology operational approach are as being means known in the art without describing.
Embodiment 1: the expression of clostridium perfringens alpha toxin albumen, purification
1, design primer
According to the alpha toxin gene order (number of logging in: AY823400 of having submitted on Genebank, its sequence is as shown in Seq ID No:1) amplification alpha toxin part specific gene fragment, design primer: primer1 and primer2, and add respectively Nco I and BamH I restriction enzyme site;
primer1：5'-AGGGATGGAAAGATTGATGG-3' Nco I Seq ID No:3
primer2：5'-TTATTTTATATTATAAGTTGAATTTCCTGA-3' BamH I Seq ID No:4
2, preparation template
Be inoculated in a blood plate culture medium (component: after 100 ml distilled waters, 1 gram of glucose and the sterilizing of 3.8 grams of bean agar powders with inoculating loop picking A type bacillus perfringens strain, adding 5 milliliters of Sheep Bloods to divide installs on six blood plates) upper, 37 ℃ of anaerobism (88%N
2, 7%H
2, 5%CO
2) cultivation 36h.Containing 7-8 milliliter THIOGLYCOLLIC ACID salt broth (FT, medium component is general) in vitro 37 ℃ of anaerobism are cultivated 12h in one to get single colony inoculation, and the culture fluid DNA extraction test kit (the precious biotech firm in Dalian) of getting 1～5ml extracts DNA profiling.
3, pcr amplification and recovery genes of interest
(1) amplifying target genes
The masterplate DNA of PCR reaction system (25ul): 1.0ul, 10 × PCR buffer (Mg
2+free) 2.5ul, MgCl
2(2.5mmol/L) 2ul, dNTP(2.5mmol/L) 1.3ul, the each 1ul of upstream and downstream primer (25 μ mol/L), Taq archaeal dna polymerase (5U/ μ L) 0.2ul; Finally supply 25ul with sterilizing distilled water.
PCR response procedures: 94 ℃ of 5min, 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 70s, totally 30 circulations are finally extended 10min in 72 ℃.
(2) reclaim genes of interest band
Pcr amplification product is after agarose gel electrophoresis, and DNA gel reclaims test kit and reclaims genes of interest band, and concrete steps are as follows:
1. under uviol lamp, cut out the agarose gel that contains target DNA, exhaust the liquid of gel surface with napkin.Now should notice that excision as far as possible, not containing the gel of object fragment part, reduces the volume of gel as far as possible, improve the response rate of DNA.
2. shred gel piece, to improve the response rate of gel, weigh the weight of gel piece, the volume of calculated for gel piece, calculates with 1mg=1ul.
3. in gel piece, add equal-volume blob of viscose melting liquid DR-I buffer.
4., after evenly mixing, 75 ℃ of heating and melting blob of viscoses, are now interrupted concussion and mix, and make blob of viscose fully melt 6-10min.
5. in above-mentioned blob of viscose melting liquid, add again the 3. DR-II buffer of described DR-I buffer1/2 volume of step, evenly mix.
6. solution obtained above is transferred in adsorption column, the centrifugal 1min of 12000rpm, discards filtrate.
7. the RinseA of 500ul is added step 6. in adsorption column after treatment, the centrifugal 30s of 12000rpm, discards filtrate again.
8. 700ulRinseB is added 7. in adsorption column after treatment, the centrifugal 30s of 12000rpm, discards filtrate again.
9. 700ulRinseB is added 8. in adsorption column after treatment, the centrifugal 30s of 12000rpm, discards filtrate again.
10. adsorption column is placed on the centrifuge tube of a 1.5ml, adds the sterile purified water of 25ul in the centre of adsorption column film, room temperature leaves standstill 1min, the centrifugal 1min of 12000rpm, eluting target DNA.
(3) alpha toxin target DNA is cloned into pMD18-T Vector carrier
By the target DNA of eluting in step (2) with pMD18-T Vector(purchased from Beijing Tian Gen biochemical technology company limited) mix, system is: pMD18-T Vector1ul, recovery product (being the target DNA of eluting) 3ul, ddH
2o1ul, solution I5ul, connects 5-7h in 16 ℃ after mixing.To connect after product mixes with Top10 competent cell (being century bio tech ltd purchased from Beijing health) and transform, screening recombinant conversion bacterium is extracted plasmid.
It is described that by connecting product, mix the concrete steps that transform with Top10 competent cell as follows:
1. get a centrifuge tube, add the Top10 competent cell melting on 50ul ice bath, then add connection product 10ul, mix gently, in ice bath, place 30min.
2. heat shock 30s in 42 ℃ of water-baths, then transfers to centrifuge tube 2min in ice bath fast, and this process is not shaken centrifuge tube.
3. add the aseptic LB fluid medium of 500ul (100mlLB fluid medium component: Tryptone1ml, Yeast extract0.5ml, Nacl1ml) again, mix and be placed on 37 ℃, 200r/min cultivates 1h.
Described screening recombinant conversion bacterium is extracted plasmid, and concrete steps are as follows:
The competent cell having transformed in the upper step of absorption 200ul is added to and contains the antibiotic LB agar culture medium of 100ug/ml ammonia benzyl (plain agar culture medium) (100mlLB agar culture medium component: Tryptone1ml, Yeast extract0.5ml, Nacl1ml, 1.5g agar powder) upper, cell is evenly coated with and is opened.Flat board is placed in to 37 ℃ and is absorbed to liquid, be inverted dull and stereotyped, 37 ℃ of incubated overnight.On the flat board of 37 ℃ of incubated overnight, sterile working's picking list colony inoculation is in containing the antibiotic LB fluid medium of 100ug/ml ammonia benzyl (100mlLB fluid medium component: Tryptone1ml, Yeast extract0.5ml, NaCl1ml) increase bacterium, 37 ℃ of shaken cultivation 12-14h, according to plasmid extraction kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) explanation, extract plasmid, concrete steps are as follows:
1. column equilibration step: to the balance liquid BL that adds 500 μ l in adsorption column CP3,12000rpm, centrifugal 1 minute, outwell the waste liquid in collecting pipe, adsorption column is relay and reclaimed in collector.
2. the bacterium liquid of getting 5ml incubated overnight adds in centrifuge tube, uses conventional desk centrifuge, and 12000rpm centrifugal 1 minute, absorbs supernatant as far as possible.
3. add 250 μ l solution P1 to leaving in the centrifuge tube of bacterial sediment again, use the pipettor precipitation that thoroughly suspends.
4. in centrifuge tube, add 250 μ l solution P2 again, gentle spinning upside down makes its abundant cracking for 6-8 time.
5. in centrifuge tube, add 350 μ l solution P3 again, spinning upside down 6-8 time of gentleness, fully mixes immediately, now occurs white flocculent deposit, 12000rpm, and centrifugal 10 minutes, there is precipitation in centrifuge tube bottom now.
6. the supernatant of previous step being collected is transferred in adsorption column CP3 with pipettor, and 12000rpm, outwells the waste liquid in collecting pipe at centrifugal 30-60 second, and adsorption column CP3 is put into collecting pipe.
7. in adsorption column CP3, add 600 μ l rinsing liquid PW again, 12000rpm, centrifugal 30-60 second, outwells the waste liquid in collecting pipe, and adsorption column CP3 is put into collecting pipe.
8. repetitive operation step 7..
9. adsorption column CP3 is put into collecting pipe, 12000rpm, centrifugal 2 minutes.
10. adsorption column CP3 is placed in to a clean centrifuge tube again, drips 50-100 μ l elution buffer EB to the middle part of adsorbed film, room temperature is placed 2 minutes, and 12000rpm, collects plasmid solution in centrifuge tube for centrifugal 2 minutes.
Described balance liquid BL, solution P1, solution P2, solution P3, rinsing liquid PW, adsorption column CP3, elution buffer EB are the component content in plasmid extraction kit.
By 37 ℃ of enzyme action 3h of the restricted enzyme for plasmid DNA obtaining (Nco I and BamH I), enzyme action system 10ul: the each 0.5ul of restricted enzyme (Nco I and BamH I), plasmid 6ul, 10 × buffer1ul, ddH
2o2ul.Enzyme action product is observed enzyme action segment in 1% agarose gel, determines positive recombiant plasmid, send Jin Site Engineering Co., Ltd to carry out nucleotide sequencing.
4, build prokaryotic expression carrier pET28a-α
By positive sequencing result α clone's recombiant plasmid and expression vector pet28a(purchased from Beijing Tian Gen biochemical technology company limited) all use Nco I and BamH I double digestion, hatch 3h for 37 ℃.Enzyme action system 50ul: the each 2.5ul of restricted enzyme, plasmid 30ul, 10 × buffer5ul, ddH
2o10ul.
Enzyme action product, through 1% agarose gel electrophoresis, reclaims object band and pet28a linear carrier, and concrete recycling step reclaims test kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.) explanation with DNA gel.
To reclaim product and be connected with pet28a linear carrier, system is: pet28a linear carrier 1ul, genes of interest fragment (reclaiming product) 3ul, ddH
2o1ul, solution I5ul, connects 5-7h in 16 ℃ after mixing.To connect product and transform after mixing with BL21 (DE3) pLysS competent cell (purchased from Beijing Chinese mugwort beguine Bioisystech Co., Ltd) (prokaryotic expression host), screening recombinant conversion bacterium is extracted plasmid, and concrete steps are as follows:
1. get on ice bath and melt BL21 (DE3) pLysS competent cell 50ul, add and connect product 10ul, mix gently, in ice bath, place 30min.
2. heat shock 45s in 42 ℃ of water-baths, then transfers to pipe 2min in ice bath fast, and this process is not shaken centrifuge tube.
3. in centrifuge tube, add the aseptic LB fluid medium of 500ul, mix and be placed on 37 ℃, 200 turn/min cultivate 1h.
4. draw competent cell that 200ul transformed and be added to and contain on the antibiotic LB agar culture medium of 100ug/ml Kana, cell is evenly coated with and is opened.Flat board is placed in to 37 ℃ and is absorbed to liquid, be inverted dull and stereotyped, 37 ℃ of incubated overnight.On the flat board of 37 ℃ of incubated overnight, sterile working's picking list colony inoculation is in increasing bacterium containing the antibiotic LB fluid medium of 100ug/ml Kana, and 37 ℃ of shaken cultivation 12-14h, obtain recombinant bacterial strain.
Recombinant bacterial strain is illustrated and extracts plasmid according to plasmid extraction kit (purchased from TIANGEN Biotech (Beijing) Co., Ltd.).Nco I and 37 ℃ of enzyme action 3h of BamH I restricted enzyme for the plasmid DNA that extraction obtains, enzyme action system 10ul: the each 0.5ul of restricted enzyme, plasmid 6ul, 10 × buffer1ul, ddH
2o2ul.Enzyme action product is observed enzyme action segment in 1% agarose gel, determines positive recombiant plasmid pET28a-α, send Jin Site Engineering Co., Ltd to carry out nucleotide sequencing.The homology that Blast compares surveyed alpha toxin complete genome sequence online and GenBank has delivered corresponding sequence is more than 98%.
Order as BL21 (DE3)-α through the definite recombinant bacterial strain that has proceeded to positive recombiant plasmid pET28a-α of enzyme action order-checking above-mentioned.
5, the abduction delivering of alpha toxin albumen, purification and specific detection
BL21 obtained above (DE3)-α is inoculated in the LB fluid medium containing 100ug/ml Kana antibiotic (bio tech ltd is flown upward in Tai'an), 37 ℃ of concussions are cultivated, in the time that bacterium liquid OD value reaches 0.6, the IPTG that interpolation final concentration is 0.1mM, 37 ℃, abduction delivering 12h.
By the bacterium liquid centrifugal collecting precipitation of abduction delivering, add 0.01M PBS solution resuspended, ultrasonic degradation cell (ultrasonic degradation program: 1s, 1s, 20min, 25 ℃), bacterium liquid centrifugal (8000rpm/min, 15min, 4 ℃) after cracking completely, separates supernatant 1 for subsequent use.0.01M PBS solution rinsing 2 times for precipitation, the centrifugal supernatant 2 of abandoning, by the resuspended precipitation of urea liquid (Sheng Ruitai Science and Technology Ltd. in Beijing), precipitation after resuspended with separate first the supernatant 1 that obtains respectively by Ni-Agarose His label protein purification column (Nanjing Genscript Biotechnology Co., Ltd.) purification, obtain alpha toxin albumen.
Sample after purification carries out SDS-PAGE electrophoresis, and detects the specificity of expressing protein by Western blot.
Embodiment 2: alpha toxin albumen toxicity test
By the culture supernatant of BL21 (DE3)-α (being supernatant 1), bacterium liquid in thalline and cellular lysate thing (step 5) after cracking completely) and the strong malicious A528 culture supernatant of bacillus perfringens be seeded in respectively egg yolk agar flat board and (take agar culture medium finished product 50g, add deionized water 1L, agitating heating is boiled to dissolving completely, subpackage triangular flask, 121 ℃ of sterilizing 15min, be chilled to 50 ℃ of left and right, every 100mL culture medium adds 50% the outstanding 10-15mL of aseptic yolk saline, shake up and pour into immediately six plates, solidify) on, 36 ℃ of anaerobism are cultivated 24h, the culture supernatant of BL21 (DE3)-α, all there is not the muddy ring of characteristic in thalline and cellular lysate thing, and there is the muddy ring of milky in the strong malicious A528 culture supernatant of bacillus perfringens, illustrate that BL21 (DE3)-α has not possessed the phospholipase C activity of alpha toxin, through the balb/c white mice (18-20g, 10) of lumbar injection BL21 (DE3)-α, all survivals after 3 weeks, every white mice is all without clinical symptoms.
The strong malicious A528 culture supernatant preparation method of described bacillus perfringens: NCTC528 is inoculated in a blood plate culture medium (after 100 ml distilled waters, 1 gram of glucose and the sterilizing of 3.8 grams of bean agar powders with inoculating loop picking A type bacillus perfringens strain, adding 5 milliliters of Sheep Bloods to divide installs on six blood plates) upper, 37 ℃ of anaerobism (88%N
2, 7%H
2, 5%CO
2) cultivation 36h.Containing 7-8 milliliter THIOGLYCOLLIC ACID salt broth (FT) (medium component is general) in vitro 37 ℃ of anaerobism are cultivated 12h, obtain bacillus perfringens A528 bacterial culture fluid in one to get single colony inoculation, and the centrifugal 30s of 12000rpm, gets supernatant and get final product.
Embodiment 3: alpha toxin recombinant vaccine safety verification
With 4 of the rabbit of body weight 1.5-2 kilogram, 5 milliliters of each intramuscular injection vaccines, observe 10, all strong living, injection site does not necrose.
Embodiment 4: alpha toxin recombinant vaccine steriling test and thimerosal assay
Carry out asepsis growth with reference to 301 pages of " People's Republic of China's veterinary biologics quality standard " (calendar year 2001 version) appendix.
Carry out with reference to 313 pages of " People's Republic of China's veterinary biologics quality standard " (calendar year 2001 version) appendix, meet " pertinent regulations of goods check " (thimerosal content is no more than 0.01%).
Embodiment 5: alpha toxin recombinant vaccine immunizing dose research
(1) get 24 of 1.5-2kg healthy rabbits, be divided into matched group and experimental group, every group each 12, the alpha toxin gene engineering vaccine of experimental group every intramuscular injection 1ml, 2ml, 3ml, every group of 4 repetitions, matched group is intramuscular injection 1ml, 2ml, 3ml physiological saline solution respectively, every group of 4 repetitions, after 21 days, 24 rabbits intravenous injection 1ml A type bacillus perfringens respectively, observe 3-5 day, 12 rabbits of matched group are all dead.In immune group there is lassitude in the rabbit of intramuscular injection 1ml alpha toxin recombinant vaccine, within 2nd, occurs afterwards death, and ill symptoms does not all appear in intramuscular injection 2ml, 3ml experimental group.Determine that 2ml vaccine is as the conventional dosage of inoculation of rabbit.
(2) get 40 of 1.5kg left and right chickens, be divided into matched group and experimental group, every group each 20, the alpha toxin gene engineering vaccine of experimental group every intramuscular injection 0.1ml, 0.25ml, 0.5ml, 0.75ml, every group of 5 repetitions, matched group is subcutaneous injection 0.1ml, 0.25ml, 0.5ml, 0.75ml physiological saline solution respectively, every group of 5 repetitions.After 21 days, 40 chickens intramuscular injection 1ml A type bacillus perfringens respectively, observe 3-5 day, 20 chickens of matched group are all dead, dissect the symptom of the necrotic enteritis such as the serious intestinal tympanites of appearance and gangrenosum acne are hemorrhage, and in immune group, lassitude appears in the chicken of subcutaneous injection 0.1ml, 0.25ml alpha toxin recombinant vaccine, in death on the 2nd, dissect and find its intestinal tympanites, the symptom of dying of illness such as hemorrhage, subcutaneous injection 0.5ml, 0.75ml experimental group are all healthy, after dissection without bad phenomenon.Determine that 0.5ml vaccine is as the conventional dosage of inoculation of chicken.
Claims (3)
1. a clostridium perfringens alpha toxin recombinant vaccine, is characterized in that clostridium perfringens alpha toxin albumen and the aluminum hydroxide adjuvant ratio take volume ratio as 9:1 carries out emulsifying, adds 0.01 gram of thimerosal to obtain in every 100ml emulsion;
The preparation process of described clostridium perfringens alpha toxin albumen is as follows:
1) design primer
According to the alpha toxin gene order amplification alpha toxin specific gene fragment of having submitted on Genebank, design primer: primer1 and primer2, and add respectively Nco I and BamH I restriction enzyme site;
primer1：5'-ATGAAAAGAAAGATTTGTAAGGCG-3' Nco I
primer2：5'-TTATTTTATATTATAAGTTGAATTTCCTGAAATCC-3' BamH I
2) prepare template and pcr amplification genes of interest
With inoculating loop picking A type bacillus perfringens strain, be inoculated in a blood plate culture medium, 37 ℃ of anaerobism are cultivated 36h; Get single colony inoculation and contain 7-8 milliliter THIOGLYCOLLIC ACID salt broth in vitro, 37 ℃ of anaerobism cultivation 12h, cultivation bacterium liquid extraction DNA profiling in one; Pcr amplification genes of interest, amplified production, after agarose gel electrophoresis, reclaims amplified production;
3) alpha toxin target DNA is cloned into pMD18-T Vector carrier
By step 2) in the extension amplification outcome that reclaims to pMD18-T Vector, after clone products is mixed with Top10 competent cell, transform, screen recombinant conversion bacterium extraction plasmid pMD18-T-α; Determine positive recombiant plasmid pMD18-T-α through enzyme action order-checking;
4) build prokaryotic expression carrier pET28a-α
By positive recombiant plasmid pMD18-T-α and expression vector pet28a double digestion, enzyme action product, through 1% agarose gel electrophoresis, reclaims object band and pet28a linear carrier; To reclaim product and be connected with pet28a linear carrier, build prokaryotic expression carrier pET28a-α; After pET28a-α is mixed with BL21 (DE3) pLysS competent cell, transform, obtain recombinant bacterial strain BL21 (DE3)-α;
5) abduction delivering of alpha toxin albumen, purification
With the IPTG of 0.1mM, 37 ℃, to recombinant bacterium abduction delivering 12h, the purified alpha toxin albumen that obtains.
2. a preparation method for clostridium perfringens alpha toxin recombinant vaccine, is characterized in that comprising the steps:
1) design primer
According to the part specific gene fragment of the alpha toxin gene order amplification alpha toxin of having submitted on Genebank, design primer: primer1 and primer2, and add respectively Nco I and BamH I restriction enzyme site;
primer1：5'-AGGGATGGAAAGATTGATGG-3' Nco I
primer2：5'-TTATTTTATATTATAAGTTGAATTTCCTG A-3' BamH I
2) prepare template and pcr amplification genes of interest
With inoculating loop picking A type bacillus perfringens strain, be inoculated in a blood plate culture medium, 37 ℃ of anaerobism are cultivated 36h; Get single colony inoculation and contain 7-8 milliliter THIOGLYCOLLIC ACID salt broth in vitro, 37 ℃ of anaerobism cultivation 12h, culture fluid extraction DNA profiling in one; Pcr amplification genes of interest, amplified production, after agarose gel electrophoresis, reclaims amplified production;
3) alpha toxin target DNA is cloned into pMD18-T Vector carrier
By step 2) in the extension amplification outcome that reclaims to pMD18-T Vector, after clone products is mixed with Top10 competent cell, transform, screen recombinant conversion bacterium extraction plasmid pMD18-T-α; Determine positive recombiant plasmid pMD18-T-α through enzyme action order-checking;
4) build prokaryotic expression carrier pET28a-α
By positive recombiant plasmid pMD18-T-α and expression vector pet28a double digestion, enzyme action product, through 1% agarose gel electrophoresis, reclaims object band and pet28a linear carrier; To reclaim product and be connected with pet28a linear carrier, build prokaryotic expression carrier pET28a-α; After pET28a-α is mixed with BL21 (DE3) pLysS competent cell, transform, obtain recombinant bacterial strain BL21 (DE3)-α;
5) abduction delivering of alpha toxin albumen, purification
With the IPTG of 0.1mM, 37 ℃, to recombinant bacterium abduction delivering 12h, the purified alpha toxin albumen that obtains.
6) by described alpha toxin albumen and aluminum hydroxide adjuvant, the ratio take volume ratio as 9:1 is carried out emulsifying, adds 0.01 gram of thimerosal to obtain clostridium perfringens alpha toxin recombinant vaccine in every 100ml emulsion.
Clostridium perfringens alpha toxin recombinant vaccine as claimed in claim 1 prevention because of the application in the caused animal necrotic enteritis of A type bacillus perfringens, enterotoxemia and gangrene after trauma.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109602898A (en) * | 2018-12-28 | 2019-04-12 | 江苏省农业科学院 | A kind of clostridium perfringens alpha toxin recombinant vaccine and preparation method thereof |
| CN114107304A (en) * | 2021-12-09 | 2022-03-01 | 佛山市正典生物技术有限公司 | Recombinant coccidian vector for expressing alpha toxin protein and fluorescent label protein and detection method thereof |
| CN115011534A (en) * | 2022-03-23 | 2022-09-06 | 山东农业大学 | Mutant strain of rhizoma nitrogen-fixing rhizobium ORS571, construction method and application |
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| CN101198352A (en) * | 2005-04-18 | 2008-06-11 | 谢尔英·普劳有限公司 | C. perfringens alpha toxoid vaccine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109602898A (en) * | 2018-12-28 | 2019-04-12 | 江苏省农业科学院 | A kind of clostridium perfringens alpha toxin recombinant vaccine and preparation method thereof |
| CN109602898B (en) * | 2018-12-28 | 2022-02-22 | 江苏省农业科学院 | Clostridium perfringens alpha toxin gene engineering vaccine and preparation method thereof |
| CN114107304A (en) * | 2021-12-09 | 2022-03-01 | 佛山市正典生物技术有限公司 | Recombinant coccidian vector for expressing alpha toxin protein and fluorescent label protein and detection method thereof |
| CN114107304B (en) * | 2021-12-09 | 2024-02-02 | 佛山市正典生物技术有限公司 | Recombinant coccidium vector for expressing alpha toxin protein and fluorescent tag protein and detection method thereof |
| CN115011534A (en) * | 2022-03-23 | 2022-09-06 | 山东农业大学 | Mutant strain of rhizoma nitrogen-fixing rhizobium ORS571, construction method and application |
| CN115011534B (en) * | 2022-03-23 | 2023-11-03 | 山东农业大学 | Mutant strain of rhizobium azotembotryococcus ORS571, construction method and application |
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