CN101912604B - Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof - Google Patents
Preparation method of live vector vaccine for controlling chicken coccidiosis and application thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of a live vector vaccine for controlling chicken coccidiosis, comprising the following steps of obtaining a recombinant expression vector by connecting a mature peptide of an eimeria tenella sporozoite antigen gene 3-1E gene and an expression vector of bacillus subtilis, obtaining a recombinant strain by transferring the recombinant expression vector into a bacillus subtilis host, and culturing the recombinant strain to finally obtain the live vector vaccine for controlling the chicken coccidiosis by. The live vector vaccine can be used for controlling and/or treating the chicken coccidiosis. The invention also discloses an oral biological preparation for controlling the chicken coccidiosis, prepared by utilizing the live vector vaccine. The oral biological preparation comprises the live vector vaccine and a vector. The oral biological preparation per gram contains the live vector vaccine with a living bacteria number of 108-109cfu.
Description
Technical field
The present invention provides a kind of preparation method of live vector vaccine with control chicken coccidiosis and uses thereof.
Background technology
Chicken coccidiosis is a kind of to the very big protozoacide of poultry husbandry harm; The intensification chicken farm is the optimum place of coccidiosis outburst; The infection of 78.7% intensification chicken farm various degrees, annual about 2,000,000,000 pounds (Lee, 2010) of economic loss that the whole world causes because of chicken coccidiosis.Chicken coccidiosis is mainly infected by one or more Eimerias and causes, and mainly parasitizes caused a kind of parasitic protozoa disease in the Intestinum Gallus domesticus tract epithelial cell by the Eimeria coccidiosis; Be mainly in for 3 monthly ages with interior chick, especially with the easy infection of the chickling of 15 ages in days～50 ages in days; The month of warm moist is multiple, and winter, morbidity was less.Suffer from the acute coccidiosis, the course of disease a few days to 2～3 weeks, the 6～10d death of most chickens in the morbidity back, the chickling mortality rate is 50%.The chronic type pilosity is born in for 4～June, becomes the fowl disease journey slow, and mortality rate is lower.Its sickness rate can reach 100%, and case fatality rate is 20%～30%, and severe patient can be up to 80%.
The environmental health of doing hen house well is the key link of anti-system coccidiosis, and chemoprophylaxis is the important means of anti-system coccidiosis.Since Levine finds that sulfa drugs has the anticoccidial effect, kind more than 50 has been arranged as the medicine that prevents coccidiosis.Roughly be divided into three major types by its chemical constitution and production process: one type is the polyethers ion carrier antibiotic, and another kind of is chemosynthesis class anticoccidial drug, and the 3rd type is Chinese herbal and crude drugs preparations.Because chemicals control coccidiosis exists Drug resistance, the improper deficiency that remains with shortcoming such as drug residue and immunoprophylaxis of administrated method, people are attempting the exploitation Chinese herbal medicine resource; In order to the control chicken coccidiosis; Some researchs show, but Chinese herbal medicine not only has the effect of the worm of killing, and can also strengthen the immunologic function of chicken body; Promote the growth promoter of chicken, improve rate of body weight gain and fertility performance.At present research and utilization with developed multiple Chinese herbal medicine anticoccidiosis medicine, control coccidiosis effect is (Jin Ailan, 2009, the red and Liu Yi of Li Xu, 2009) obviously.And for example patent 200910304688.1 discloses a kind of Chinese medicine composition of treating fowl parasitic coccidiosis, and that its compositions absorbs is fast, safety coefficient is high, no drug residue, has no side effect, and can not develop immunity to drugs.But, still be difficult to see the dawn of breakthrough because China's Chinese herbal medicine exists extraction complicacy, the restriction of research degree and the deficiency of investigative technique, input etc. at present.
Use strong malicious Seedling and cause the weak coccidiosis Seedling a kind of means of can yet be regarded as that inoculate against, strong malicious Seedling is only applicable to kind of a chicken, not exclusively is applicable to laying hen and broiler, though broiler available a little less than malicious coccidiosis Seedling, just have poor stability again, inoculum concentration is restive, problems such as expense height.The coccidiosis cell surface antigen directly contacts with host cell, participates in coccidiosis and invades host cell and subsequent internalization process, and excite host's immunoreation as the major antigen composition, so be the ideal candidate antigens of development recombiant vaccine.Sporozoite surface antigen is meant the antigen of in the Sporogony process, expressing, and studies more 3-1E, cSZ1 (Qin Zonghua, 2005), the cSZ2 (Shah of mainly containing; 2010), TA4 (Xu, 2008, Zhang Li; 2006) etc., wherein 3-1E all has expression at zygoblast and merozoite stage.Disclose 3-1E gene of utilizing the chicken coccidiosis etc. like patent 200510108341.1 and made up fusion gene and chimeric, and prepared fusion rotein; The cDNA that Song etc. (2000) pile type Eimeria (E.acervulina) 3-1E antigen gene with chicken inserts carrier pMP13, is built into dna vaccination, and E.acervulina detects higher circulating antibody level (Lillehoj, 2000) after attacking worm.Lillehoj etc. (2005) are connected the 3-1E gene respectively with cytokine adjuvants such as interleukin I L-1, IL-2, IL-6, IL-8, IL-18 and gamma interferons, recombinant has tangible immanoprotection action (Lillehoj, 2005).Qin Zonghua etc. (2005) are on the basis of successfully cloning E.acervulina Guangdong strain (GD) zygoblast surface protection property antigen gene cSZ1; Made up the recombinant attenuated Salmonella of expressing cSZ1; Then with 108 or 109CFU/ peroral immunity 4 Japanese instar chicklings, 2 secretions that test group all can be induced intestinal specificity IgA during 25 ages in days; And can reduce by 25.1% and 46.4% egg capsule generation (Qin Zonghua, Xie Mingquan, 2005) respectively; Similar is, Shah etc. are with cSZ2 constructed dna vaccine, and the result shows that the cSZ2DNA vaccine can the induction of immunity reaction, and through reducing the intestinal infringement, the scale of construction alleviates with oocyst rate and formed the protection (Shah, Yan, 2010) of district portion.Xu etc. (2006) form mosaic gene (prolE) with E.tenella 3-1E antigen gene and chicken interferon-gamma gene fusion; Chimeric DNA vaccine and chimeric protein (rlE) have been made up; Research shows and dna vaccination (proE); Dna vaccination (prolE) can reduce egg capsule to a greater extent and come off, but dna vaccination (prolE) fails to increase antibody titer and body weight; But chimeric protein (rlE) then reduces egg capsule to a greater extent to come off, and also can increase antibody titer and body weight (Xu, 2006) simultaneously.It is thus clear that the immanoprotection action of coccidiosis more and more receives people's attention, especially with the recombinant biological agent best results of zygoblast antigen or gene.
Yet in large-scale cultivation, recombiant protein protection effect is unsatisfactory, by oral route especially, and antigen causes the deficiency of protection dosage in reasons such as digestive tract degraded, absorbance are low.
The disclosed production vaccine of above document and patent has adopted in attenuated vaccine, dna vaccination, recombiant protein and the agent of medium-height grass recurrence due to taking drug and has improved the immanoprotection action of chicken.And utilize attenuated vaccine, and there is virulence to return strong danger, antibody cannot not held time longly, and the vaccine that has has certain side effect and is unfavorable for the transportation preservation because attenuation is not thorough; Dna vaccination produces safety issue, comprises that possibility is carcinogenic and produces dna antibody and genetic drift etc.; There is complex manufacturing process in recombiant protein, cost is higher and vaccine needs shortcomings such as cryopreservation; And there are defectives such as this height of deduction, mechanism of action complicacy in Chinese herbal medicine.
The list of references of mentioning in the preceding text is specific as follows:
1.Lee, B.H., W.H.Kim, J.Jeong, J.Yoo; Y.K.Kwon, B.Y.Jung, J.H.Kwon, H.S.Lillehoj, and W.Min; Prevalence and Cross-Immunity of Eimeria Species on Korean Chicken Farms.J Vet Med Sci, 2010. (L ee, B.H., W.H.Kim, J.Jeong; J.Yoo, Y.K.Kwon, B.Y.Jung, J.H.Kwon; H.S.Lillehoj, and W.Min, different Amy coccuses are in the popular of Korea S's chicken house and cross immunity .J Vet Med Sci, 2010.)
2. Jin Ailan, Chen Xiuzhen opens and supplies neck, and Li Yafeng is permitted to rise, ten thousand pairs of shows of and, Chinese medicine Radix Dichroae and prescription thereof and Western medicine are to the curative effect contrast test of chicken coccidiosis. Chinese animal quarantine, 2009 (008): p.56-57.
3. the red and Liu Yi of Li Xu, the anti-chicken coccidiosis of Chinese herbal compound preparation Research on effect. Hunan agricultural sciences, 2009 (005): p.146-148.
4. deep ancestor is magnificent, Xie Mingquan, and Cai Jianping, Ahmed, and leaf show China, heap type Eimeria cSZ1 gene is in the expression and the immune protective effect research of attenuation salmonella. parasite and medical insect journal, 2005.12 (001): p.6-13.
5.Shah, M.A., R.Yan; L.Xu, X.Song, and X.Li; A recombinant DNA vaccine encodingEimeria acervulina cSZ-2 induces immunity against experimental E.tenella infection.VetParasitol, 2010.169 (1-2): p.185-9. (Shah, M.A.; R.Yan, L.Xu, X.Song; And X.Li, the recombinant DNA vaccine induction of immunity opposing Eimeria tenella of coding heap shape Eimeria cSZ-2 is infected .Vet Parasitol, 2010.169 (1-2): p.185-9.)
6.Xu, Q., X.Song; L.Xu, R.Yan, M.A.A.Shah; And X.Li, Vaccination of chickens witha chimeric DNA vaccine encoding Eimeria tenella TA4 and chicken IL-2 induces protectiveimmunity against coccidiosis.Veterinary parasitology, 2008.156 (3-4): (Xu p.319-323.; Q., X.Song, L.Xu; R.Yan, M.A.A.Shah, and X.Li; The chicken of the chimeric DNA vaccine of inoculation coding Eimeria tenella TA4 and chicken IL-2 is induced protection immunity opposing coccidiosis .Veterinary parasitology, 2008.156 (3-4): p.319-323.)
7. open jasmine, Qin Zerong, Cui Shangjin, He Zhaoqing, and Liu Shang is high, the cloning and expression of Eimeria tenella TA4 antigen gene cDNA in lactococcus lactis. Chinese Preventive Veterinary Medicine newspaper, 2006.28 (003): p.271-274.
8.Lillehoj, H.S., K.D.Choi, M.C.Jenkins; V.N.Vakharia, K.D.Song, J.Y.Han; And E.P.Lillehoj, A recombinant Eimeria protein inducing interferon-gamma production:comparison ofdifferent gene expression systems and immunization strategies for vaccination againstcoccidiosis.Avian Dis, 2000.44 (2): (Lillehoj p.379-89.; H.S., K.D.Choi, M.C.Jenkins; V.N.Vakharia, K.D.Song, J.Y.Han; And E.P.Lillehoj, the protein induced Lambda interferon of reorganization Amy coccus produces: relatively vaccine is resisted coccus sick different genes expression system and immunization strategy .Avian Dis, 2000.44 (2): p.379-89.)
9.Lillehoj, H.S., X.Ding; R.A.Dalloul, T.Sato, A.Yasuda; And E.P.Lillehoj, Embryovaccination against Eimeria tenella and E. acervulina infections using recombinant proteins and cytokine adjuvants.J Parasitol, 2005.91 (3): (Lillehoj p.666-73.; H.S., X.Ding, R.A.Dalloul; T.Sato, A.Yasuda, and E.P.Lillehoj; Embryo. utilize embryo's vaccination opposing Eimeria tenella of recombiant protein and cytokine adjuvant to infect .J Parasitol, 2005.91 (3): p.666-73.) with heap shape Eimeria
10.Xu, S.Z., T.Chen; And M.Wang, Protective immunity enhanced by chimeric DNAprime-protein booster strategy against Eimeria tenella challenge.Avian Dis, 2006.50 (4): (Xu p.579-85.; S.Z., T.Chen, and M.Wang; Strengthen strategy increase protection immunity opposing Eimeria tenella through the chimeric DNA major protein and infect .Avian Dis, 2006.50 (4): p.579-85.)
Summary of the invention
The technical problem that the present invention will solve provides a kind of safe in utilization and can effectively prevent and treat the live vector vaccine and the corresponding oral biological preparation of chicken coccidiosis.
In order to solve the problems of the technologies described above; The present invention provides a kind of method for preparing of preventing and treating the live vector vaccine of chicken coccidiosis: the mature peptide of Eimeria tenella zygoblast antigen gene 3-1E gene is successively win recombinant expression carrier mutually with the expression vector of bacillus subtilis; Change recombinant expression carrier over to the bacillus subtilis host again and obtain recombinant bacterial strain; Cultivate this recombinant bacterial strain, promptly get the live vector vaccine of preventing and treating chicken coccidiosis.
As the improvement of the method for preparing of the live vector vaccine of control chicken coccidiosis of the present invention, it may further comprise the steps:
1) obtains containing the cloning vehicle of 3-1E gene, called after pMD18T-3-1E, the purpose fragment cloning of 3-1E gene is gone into carrier pMD18T vector;
2), select bacillus subtilis expression vector pBES for use;
3) obtain the enzyme action product, the 3-1E gene is got off with restricted enzyme Xba I and Pst I enzyme action from pMD18T-3-1E; Reuse restricted enzyme Xba I and Pst I with bacillus subtilis expression vector pBES enzyme action after, above-mentioned enzyme action product is connected with the bacillus subtilis expression vector pBES behind T4 dna ligase and the enzyme action; Must connect product;
4), with above-mentioned steps 3) the connection product transformed into escherichia coli Top10 competent cell of gained, single bacterium colony that picking grows shakes bacterium, carries out bacterium colony PCR checking; Obtain containing the recombiant plasmid of 3-1E, called after p BES-3-1E;
5), above-mentioned pBES-3-1E is transformed and coated plate in bacillus subtilis 1A751; Get live vector vaccine.
The present invention also provides the purposes of above-mentioned live vector vaccine simultaneously: be used to prevent and/or treat chicken coccidiosis.
The present invention also provides the oral biological preparation that utilizes the control chicken coccidiosis that above-mentioned live vector vaccine processes simultaneously, and this oral biological preparation is made up of live vector vaccine and carrier, and containing viable count in every gram oral biological preparation is 10
8Cfu～10
9The live vector vaccine of cfu.
Improvement as the oral biological preparation of control chicken coccidiosis of the present invention: carrier is that the zeolite powder of 5-50% and the corn starch of 50-95% are formed by weight ratio.Zeolite powder and corn starch all can be crossed 80～200 mesh sieves.
In the present invention, live vector vaccine can be according to method preparation provided by the invention, and zeolite powder and corn starch all can obtain through commercial mode.
The storage conditions of the oral biological preparation of control chicken coccidiosis of the present invention is: room temperature, effect duration is 2 years.
During the actual use of the oral biological preparation of control chicken coccidiosis of the present invention, add in the chicken feedstuff, can carry out feeding according to conventional method behind the uniform mixing according to 0.1%～1% the ratio that accounts for chicken feedstuff gross weight.
The present invention has made full use of the following advantage of bacillus subtilis: 1) high secretion capacity; Make recombinant expressed pharmaceutical protein not need through the purification in downstream with separate, also make excretory antigen protein effectively submission to host's immune system cause immunne response; 2) antigen protein is showed in the spore outer surface, can be used as the application of oral vaccine; 3) characteristic of heat-resisting, the acidproof and salt tolerant of spore can tolerate gastrointestinal tract environment and be more conducive to and preserve; 4) quick, simple and direct fermentation and production technology;
5) this is as probiotic bacteria, as if the effect that can play adjuvant as vaccine carrier.
Oral biological preparation provided by the present invention, have easy to use, prophylactic treatment develops simultaneously, production cost is low, characteristics such as convenient for production.
The live vector vaccine that adopts the inventive method preparation and get does not need purification, can directly be used to produce the oral biological preparation of control chicken coccidiosis, removes the complicated procedures of forming that protein is handled from, thereby has good value for applications.
The specific embodiment
Following embodiment helps those skilled in the art more fully to understand the present invention, but does not limit the present invention in any way.
Following % is weight %.
Method used among the following embodiment is conventional method if no special instructions.
Embodiment 1, contain antigen gene 3-1E construction of recombinant plasmid
One, the clone of antigen 3-1E gene
1, the clone of 3-1E gene
According to routine techniques purification spore egg capsule from the broiler feces that infects E.tenella; And mention the total RNA in the purebred spore egg capsule; With the reverse transcription test kit RNA reverse transcription is become cDNA, and be template with the cDNA of reverse transcription, with forward primer (SEQ ID NO:1):
5P
31E-BS5 '-TCTAGAAATGGGTGAAGAGGCTGATACT-3 ' and
Downstream primer (SEQ ID NO:2):
3P
31E-BS5 '-CTGCAGTTA
GTGATGGTGATGGTGATGGAAGCCGCCCTGGTACAGGT-3 ' carries out PCR, obtains genes of interest (3-1E gene).
The PCR reaction system is:
cDNA 1.0μl
Primer 5P
31E-BS(10mM) 1.0 μ l
Primer 3P
31E-BS(10mM) 1.0 μ l
Taq enzyme (5U/ μ l) 0.5 μ l
10 * PCR buffer (contains Mg
2+) 5.0 μ l
dNTP(10mM?each) 1.0μl
ddH
2O 40.5μl
Total 50.0μl
Centrifugal slightly, place the PCR appearance; PCR reaction condition: 94 ℃ of preparatory degeneration 2min; 94 ℃ of degeneration 30s, 40 ℃ of annealing 30s, 72 ℃ are extended 40s, react 5 circulations; 94 ℃ of degeneration 30s, 50 ℃ of annealing 30s, 72 ℃ are extended 40s, react 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations.The PCR product is labeled as 3-1E, and the agarose gel electrophoresis with 1% detects.And the rubber tapping recovery, the agarose gel recycling step is seen test kit (the AxyPrep dna gel reclaims test kit, AxyGEN company) explanation.
The purpose fragment cloning of the 3-1E gene (sequence is seen GenBank EF069437) of 513bp is gone among the carrier pMD18Tvector (purchasing in TaKaRa biological engineering company limited (Dalian)), obtain containing the cloning vehicle of 3-1E gene, called after pMD18T-3-1E.
2, make up bacillus subtilis expression vector pBES:
Can with reference to Fu Linglin " structure of shrimp white spot syndrome virus envelope protein VP28 hay engineering bacteria and the preparation and the disease resistance research thereof of VP28 yolk antibody. Zhejiang University's thesis for the doctorate, 2008 " make up, particular content is following:
2.1 design of primers
According to pUS186 plasmid promoter-signal peptide sequence two ends design primer, sequence is following:
5Pb18-1：5’-AGGCTTTTAAGCCGTCTGTACGTTC-3’(SEQ?ID?NO：3)
3Pb18-1：5’-AAATGCCTCACATTTGTGCCACCTA-3’(SEQ?ID?NO：4)
2.2PCR amplification promoter-signal peptide sequence
With plasmid pUS186 is template, and 5Pb18-1 and 3Pb18-1 are primer, pcr amplification length be 581bp contain B.subtilis strong promoter PE σ
43Genetic fragment with bacillus amylase natural signals peptide SP sequence.The pcr amplification condition is to get into circulation behind 94 ℃ of degeneration 2min, 94 ℃ of degeneration 45s, and 58 ℃ of renaturation 1min, 72 ℃ are extended 1min, extend 10min at 72 ℃ after 35 circulations.The PCR product is labeled as PCR/BPS, and the agarose gel electrophoresis with 1% detects.
2.3PCR product P CR/BPS purification
The agarose gel recycling step is seen test kit (the AxyPrep dna gel reclaims test kit, AxyGEN company) explanation.
2.4 double digestion PCR/BPS fragment and pBE2 carrier
Bacillus subtilis-shuttle vehicle pBE2; Be the derivative vector of pUB110, can by methods such as Guo Xinghua (Guo Xinghua, Bears accounts for; Zhou Min; Jia Shifang, Xu Yi. the structure of the multi-functional shuttle vector of bacillus subtilis one escherichia coli. biological engineering journal .1991,7 (3): 224-229) make up preservation.Respectively plasmid pBE2 and PCR product P CR/BPS are carried out EcoRI and XbaI double digestion, 37 ℃ of water-baths are spent the night.The double digestion system is distinguished as follows:
2.5 connect
In the 1.5ml centrifuge tube, add pBEC (EcoRI-XbaI) 3 μ l, PCR/BPS (EcoRI-XbaI) 5 μ l, T4DNA ligase 1 μ l, 10 * T4 DNA ligase buffer, 1 μ l, mixing gently, centrifugal slightly after, put 16 ℃ of constant temperature appearance and connect and spend the night.The connection product transformed into escherichia coli Top10 competent cell of gained, single bacterium colony that picking grows shake bacterium, carry out bacterium colony PCR checking.
Above-mentioned structure and bacillus subtilis expression vector pBES contain strong promoter and signal peptide.
3, the 3-1E gene is got off with restricted enzyme Xba I and Pst I enzyme action from pMD18T-3-1E, obtain the enzyme action product; Reuse restricted enzyme XbaI and Pst I with bacillus subtilis expression vector pBES enzyme action after, above-mentioned enzyme action product is connected (the 3-1E gene can link to each other with any hay bacterium expression vector) with the T4DNA ligase with it, the coupled reaction time is 16 hours; Must connect product.
4, with the connection product transformed into escherichia coli Top10 competent cell of above-mentioned steps 3 gained, single bacterium colony that picking grows shakes bacterium, carries out bacterium colony PCR checking, and the primer is:
Forward primer 5P
31E-BS5 '-TCTAGAAATGGGTGAAGAGGCTGATACT-3 ' and downstream primer
3P
31E-BS?5’-CTGCAGTTA
GTGATGGTGATGGTGATGGAAGCCGCCCTGGTACAGGT-3’
PCR checking system is with step 1.
The sub-upgrading grain of the segmental clone of the purpose that will increase, identify, obtain the positive cloned plasmids of the endonuclease bamhi of 513bp with the further enzyme action of restricted enzyme Xba I and Pst I.To its row analysis of checking order, obtain the correct recombiant plasmid that contains 3-1E, called after pBES-3-1E again.
The method for preparing of embodiment 2, recombined bacillus subtilis (live vector vaccine of anti-chicken coccidiosis):
1 constructed recombinant expression carrier (recombiant plasmid) pBES-3-1E transforms (can transform any bacillus subtilis strain) in bacillus subtilis 1A751 (deriving from U.S. bacillus preservation center BGSC) with the foregoing description:
Choose speckle bacillus subtilis 1A751 in 4ml GMI culture medium (test tube), 30 ℃ of overnight incubation, 100r/min (shaking slowly) 10% (volume ratio) transfers in 6ml GMI culture medium (triangular flask), cultivates 3.5h, 200r/min (shaking soon) for 37 ℃; 10% transfers in 20ml GMII culture medium (triangular flask), cultivates 90min, 100r/min (shaking slowly) for 37 ℃; Centrifugal collection thalline (the supernatant suspension thalline of 1/10 volume): 20ml bacterium liquid branch is installed to the centrifuge tube (every pipe 5ml) of 4 10ml, centrifugal collection thalline, the surplus 0.5ml supernatant of the every pipe thalline that suspends again; 0.5ml bacterium liquid adds reorganization plasmid 5 μ l; Room temperature is placed 10min;
Coated plate:
Directly inhale 200 μ l bacterium liquid coating LB (Kana 5 μ g/ml) flat board; Occur in 37 ℃ of incubators to single bacterium colony.The picking transformant is carried out PCR and enzyme action checking, identifies that clone's that amplifies 513bp is transformant.Choose speckle engineering bacteria B.subtilis (pBES-3-1E) to the LB culture medium that contains 5% (mass ratio) glucose and 0.2% (mass ratio) glutamine, 37 ℃, 180rpm shaken cultivation to cell density is 10
9Cfu/ml adsorbs with corn starch behind the collection thalline, and carries out colony counting.Specifically can be: every gram corn starch absorption 10
10The cfu live vector vaccine; Get mycopowder.
The preparation of the oral biological preparation of embodiment 3, prevention chicken coccidiosis
The zeolite powder of 25 weight portions is mixed with the corn starch of 75 weight portions mutually, and this zeolite powder and corn starch all can be crossed 100 mesh sieves; Get carrier.
The mycopowder of embodiment 2 gained is mixed by 1: 9 weight ratio with above-mentioned carrier, simply mix and promptly get the oral biological preparation of preventing and treating chicken coccidiosis; This oral biological preparation of every gram contains 10
9The live vector vaccine of cfu.
The test of embodiment 4 immunoprotections
100 1 age in days Ai Wei sound broiler are divided into 5 groups, 20 every group at random.Be respectively the non-immune matched group of non-infection (CK1), infect non-immune matched group (CK2), bacillus cereus empty carrier matched group (B.S [pBES]; Be designated as B.S-P; Its content is equal to the present invention), oral the present invention's (embodiment 3) biological preparation as test group (B.S [pBES-3-1E]; Be designated as B.S-31E), coccidiostat-decoquinate matched group (Decoquinate is designated as Dec).
Bacillus cereus empty carrier matched group: 5g bacillus cereus empty carrier/kg feedstuff.
Test group (the present invention): 5g oral biological preparation/kg feedstuff.
Coccidiostat-decoquinate matched group: 0.5g deccox/kg feedstuff.
After 7 days preliminary trial periods, test group and matched group all adopt the spice mode to carry out oral immunity, continuous immunity 10 days.Duration of test free choice feeding and drinking-water.During 17 ages in days (before attacking worm), slaughter 5 at random for every group, get whole blood, serum and cecum mucosa, in order to index determining.(attack worm after the 8th day) all slaughters during 24 ages in days.
Result of the test is following:
1, gaining effect
Table 1 is respectively organized the chicken body weight change
Notes: the different lower cases of same column shoulder mark are represented significant difference (P＜0.05), contain same letter and represent difference not remarkable (P＞0.05), down together.
Table 2 is respectively organized the chicken body weight gains and is changed
Annotate: average weight gain 1 is meant the weightening finish of attacking the preceding chicken of worm; Average weight gain 2 is meant the weightening finish of attacking chicken behind the worm;
By respectively organizing chicken body weight change difference not remarkable (P＞0.05) before table 1 and the visible immunity of table 2.Before attacking worm after the immunity, B.S-P group the relative weight gain not only is significantly higher than B.S-31E group and Dec group (P＜0.05), also is significantly higher than CK1 group (P=0.048), and the B.S-31E group is organized a little less than CK1 with the Dec group, but difference not remarkable (P＞0.05).Above result shows that the bacillus subtilis that contains empty expression vector can significantly promote the growth of broiler; The oral biological preparation group and the deccox of expressing the 3-1E gene have no adverse effects to growth of meat chicken.
After attacking worm, all infected group chicken the relative weight gain all significantly are lower than non-infected group (CK1 group) (P＜0.05); In infected group, B.S-31E group and Dec group chicken the relative weight gain are significantly higher than CK2 group and B.S-P group (P＜0.05), and B.S-31E group and Dec group difference are all not remarkable.Above result shows, the body weight that oral biological preparation group, deccox cause coccidiosis reduces has certain inhibitory action, and the bacillus subtilis that contains empty expression vector does not have this effect.
2, OPG value (gram feces egg sac number)
Check feces every day after attacking worm, collected respectively since the 5th day and respectively organize feces, collected continuously 6 days; Behind the mixing; Get 1g for every group, add the 10mL distilled water and process 10 times of diluents, after stirring, count the worm's ovum number in every gram feces with the RBC plate in microscopically.Egg capsule slip (protective rate)=[(infect non-immune group feces egg capsule output-test group feces egg capsule output)/infect non-immune group feces egg capsule output] * 100%.The result is as shown in table 3.
Table 3 is respectively organized the OPG value
Visible by table 3, there is not egg capsule in CK1 group (the non-worm group of the attacking) feces; Compare with the CK2 group, the gram feces egg capsule number average of B.S-P group, oral biological preparation group and Dec group extremely significantly reduces (P＜0.01), and wherein the oral biological preparation group extremely significantly is lower than B.S-P group and Dec group again, and the latter two differences are not remarkable.Be respectively 100% and 0% in CK1 group and CK2 group egg capsule slip, the egg capsule slip of B.S-P group, oral biological preparation group and Dec group is respectively 45.17,63.70 and 31.87%.Above result shows that the bacillus subtilis and the deccox that contain empty carrier or 3-1E antigen gene all have certain inhibitory action to the breeding in vivo of chicken coccidiosis, and wherein the effect of oral biological preparation group is best.
3, caecum lesion is kept the score
Attacked behind the worm the 8th day, and got 5 for every group and slaughter, observe caecum lesion.
Relative pathological changes score=(the non-immune group lesion score of test group lesion score/infection) * 100%
Carry out caecum lesion by the method for Johnson etc. and keep the score, caecum lesion is divided into five grades of 0-+4.
0 minute, no naked eyes pathological changes
+ 1 minute, the petechia that the caecum wall has very small amount to be dispersed in, intestinal wall does not thicken, and content is normal.
+ 2 minutes, pathological changes quantity was more, and cecal content obviously is with blood, and the caecum wall thickens slightly, and content is normal.
+ 3 minutes, volume blood is arranged in the caecum or caecum core (the caseous Fructus Musae type of blood clot or canescence block) is arranged, intestinal wall is obviously plump, and feces content is few in the caecum.
+ 4 minutes, because of being full of a large amount of blood or the enlargement of intestinal core caecum, containing the excrement slag in the intestinal core or do not contain, dead chicken also remembers+4 minutes.The result is as shown in table 4.
Table 4 is respectively organized caecum lesion and is kept the score
Visible by table 4, CK1 group caecum does not have pathological changes; Compare with the B.S-P group with the CK2 group, the caecum lesion of oral biological preparation group and Dec group is kept the score and is all extremely significantly reduced (P＜0.01), explains that oral biological preparation group and deccox all have some improvement to the caecum lesion that the chicken coccidiosis is caused.
4, anticoccidial index (ACI)
Anticoccidial index (ACI)=(survival rate+the relative weight gain rate)-(pathological changes value+egg capsule value)
Survival rate=(survival chicken number/test group chicken number during off-test) * 100%
The relative weight gain rate=(the non-immune group weightening finish of weight/non-infection when slaughtering at last) * 100%
The average lesion score (0-4) * 10 of pathological changes value=each test group
The egg capsule value is calculated (like table 5) by following method, and the result is as shown in table 6.
Table 5
The feces egg capsule is got rid of number (10
6) the egg capsule value
Less than 0.1 0
0.1-1.0 1
2.0-5.0 10
6.0-10.0 20
Greater than 11.0 40
With ACI >=180 serves as that the protection effect is remarkable, and ACI=160-179 is general for the protection effect, and ACI＜160 are the unprotect effect.
Table 6, respectively organize the anticoccidial index
Visible by table 6; All ACI that attack the worm group significantly are lower than CK1 group (P＜0.05); But Dec group and oral biological preparation group ACI have significantly improved 18.5% (P=0.002) and 18.8% (P=0.001) than the CK2 group respectively, have significantly improved 14.6% (P=0.033) and 14.9% (P=0.029) than the B.S-P group respectively.Can judge that according to " ACI >=180 are remarkable for the protection effect; ACI=160-179 is general for the protection effect; ACI＜160 are the unprotect effect " this principle the bacillus subtilis (oral biological preparation of the present invention) and the deccox of expressing the 3-1E antigen gene all have remarkable protection effect; Bacillus subtilis also has certain protective role, but effect is general.
Above presentation of results, oral biological preparation of the present invention are best biological preparation safety, easy to use of a kind of anti-prevention chicken coccidiosis effect.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (5)
1. method for preparing of preventing and treating the live vector vaccine of chicken coccidiosis; It is characterized in that: with sequence is that the Eimeria tenella zygoblast antigen gene 3-1E gene shown in the GenBank EF069437 is successively win recombinant expression carrier pBES-3-1E mutually with bacillus subtilis expression vector pBES; Change recombinant expression carrier pBES-3-1E over to bacillus subtilis 1A751 host again and obtain recombinant bacterial strain; Cultivate this recombinant bacterial strain, promptly get the live vector vaccine of preventing and treating chicken coccidiosis;
The construction method of said bacillus subtilis expression vector pBES is following:
1), design of primers
According to pUS186 plasmid promoter-signal peptide sequence two ends design primer, sequence is following:
5Pb18-1：5’-AGGCTTTTAAGCCGTCTGTACGTTC-3’
3Pb18-1：5’-AAATGCCTCACATTTGTGCCACCTA-3’；
2), pcr amplification promoter-signal peptide sequence
With plasmid pUS186 is template, and 5Pb18-1 and 3Pb18-1 are primer, pcr amplification length be 581bp contain B.subtilis strong promoter PE σ
43Genetic fragment with bacillus amylase natural signals peptide SP sequence; The pcr amplification condition is to get into circulation behind 94 ℃ of degeneration 2min, 94 ℃ of degeneration 45s, and 58 ℃ of renaturation 1min, 72 ℃ are extended 1min, extend 10min at 72 ℃ after 35 circulations; The PCR product is labeled as PCR/BPS, and the agarose gel electrophoresis with 1% detects;
3), PCR product P CR/BPS purification
The agarose gel recycling step is seen the test kit explanation, and said test kit is that the AxyPrep dna gel of AxyGEN company reclaims test kit;
4), double digestion PCR/BPS fragment and pBE2 carrier
Bacillus subtilis-shuttle vehicle pBE2 is the derivative vector of pUB110; Respectively plasmid pBE2 and PCR product P CR/BPS are carried out EcoRI and XbaI double digestion, 37 ℃ of water-baths are spent the night; The double digestion system is distinguished as follows:
10×Buffer?M?3μl
BSA?0.3μl
EcoRI?1μl
XbaI 1μl
pBE2?24.7μl
Total?30μl；
10×Buffer?M?2μl
BSA?0.2μl
EcoRI?1μl
XbaI 1μl
PCR/BPS?15.8μl
Total?20μl；
5), connect
The EcoRI of the EcoRI of the above-mentioned pBE2 carrier of adding and XbaI double digestion product 3 μ l, above-mentioned PCR/BPS and XbaI double digestion product 5 μ l, T4DNA ligase 1 μ l, 10 * T4DNA ligase buffer, 1 μ l in the 1.5ml centrifuge tube; Mixing gently; After centrifugal slightly, put 16 ℃ of constant temperature appearance connections and spend the night; The connection product transformed into escherichia coli Top10 competent cell of gained, single bacterium colony that picking grows shake bacterium, carry out bacterium colony PCR checking;
Above-mentioned structure and bacillus subtilis expression vector pBES contain strong promoter and signal peptide.
2. the method for preparing of the live vector vaccine of control chicken coccidiosis according to claim 1 is characterized in that may further comprise the steps:
1) obtains containing the cloning vehicle of 3-1E gene, called after pMD18T-3-1E, carrier pMD18T is gone in the 3-1E gene clone;
2), select bacillus subtilis expression vector pBES for use;
3) obtain the enzyme action product, the 3-1E gene is got off with restricted enzyme XbaI and Pst I enzyme action from pMD18T-3-1E; Reuse restricted enzyme XbaI and Pst I with bacillus subtilis expression vector pBES enzyme action after, above-mentioned enzyme action product is connected with the bacillus subtilis expression vector pBES behind T4DNA ligase and the enzyme action; Must connect product;
4), with above-mentioned steps 3) the connection product transformed into escherichia coli Top10 competent cell of gained, single bacterium colony that picking grows shakes bacterium, carries out bacterium colony PCR checking; Obtain containing the recombiant plasmid of 3-1E, called after pBES-3-1E;
5), above-mentioned pBES-3-1E is transformed and coated plate in bacillus subtilis 1A751; Get live vector vaccine.
3. the oral biological preparation of the control chicken coccidiosis that the live vector vaccine that utilizes method preparation according to claim 1 or claim 2 and get is processed; It is characterized in that: said oral biological preparation is made up of live vector vaccine and carrier, and it is 10 that every gram oral biological preparation contains viable count
8Cfu～10
9The live vector vaccine of cfu.
4. the oral biological preparation of control chicken coccidiosis according to claim 3 is characterized in that: said carrier is that 5～50% zeolite powder and 50～95% corn starch are formed by weight ratio.
5. the oral biological preparation of control chicken coccidiosis according to claim 4 is characterized in that: said zeolite powder and corn starch all can be crossed 80～200 mesh sieves.
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| CN102816788A (en) * | 2012-08-23 | 2012-12-12 | 东北农业大学 | Method for expressing eimeria acevulina 3-1E protein in lactococcus lactis |
| CN106267175B (en) * | 2016-09-19 | 2020-01-17 | 中国农业大学 | Chicken coccidiosis vaccine and application thereof |
| US20180153945A1 (en) * | 2016-12-05 | 2018-06-07 | The United States Of America, As Represented By The Secretary Of Agriculture | Oral e. coli vector-based vaccine for prevention of coccidiosis in poultry |
| CN107325166A (en) * | 2017-05-15 | 2017-11-07 | 华中农业大学 | Recombinant protein and its application coded by Eimeria tenella surface antigen gene SAG6 |
| CN113041343A (en) * | 2021-03-19 | 2021-06-29 | 吉林大学 | Eimeria acervulina recombinant subunit vaccine and preparation method thereof |
| CN115227810A (en) * | 2022-04-18 | 2022-10-25 | 福建农林大学 | Chicken anti-Eimeria maxima oral recombinant spore vaccine and preparation method thereof |
| CN116254214A (en) * | 2022-12-29 | 2023-06-13 | 佛山科学技术学院 | Bacillus subtilis recombinant strain and preparation method and application thereof |
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