CN101880647B - Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application - Google Patents
Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application Download PDFInfo
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Abstract
The invention belongs to the technical field of bacterial gene engineering of animals, and in particular relates to the construction of a recombinant salmonella choleraesuis strain which does not contain resistance markers and expresses major antigenic loci of porcine transmissible gastroenteritis virus, vaccine preparation and application. In the recombinant salmonella choleraesuis strain C500 (pYA-2SLN) which does not contain the resistance markers and expresses the major antigenic loci of the porcine transmissible gastroenteritis virus, the preservation No. is CCTCC NO: M 209189, and the strain misses asd genes which are necessary for the growth of salmonella choleraesuis and contains plasmid capable of expressing the asd genes, genes of an antigenic locus A, an antigenic locus D and an antigenic locus N321 of the porcine transmissible gastroenteritis virus in the strain. The invention also discloses a method for preparing the salmonella choleraesuis and porcine transmissible gastroenteritis vaccine by utilizing the recombinant strain and application thereof. The prepared recombinant vaccine can stimulate pigs to generate protective immune response for resisting the salmonella choleraesuis and the porcine transmissible gastroenteritis virus, and prevent the infection of the salmonella choleraesuis and the porcine transmissible gastroenteritis virus effectively.
Description
Technical field
The invention belongs to the animal bacteria gene engineering technology field, be specifically related to the N in A, D antigen site and the N albumen in a kind of expression pig infectious gastroenteritis virus S protein that does not contain resistance marker
321The structure and the application of the recombinant salmonella choleraesuis living vaccine bacterial strain of antigen site gene.
Background technology
Salmonella choleraesuls (Salmonella choleraesuis) are the main pathogen that causes 2-4 monthly age necrotic enteritis; Cause after the infected pigs with classical symptoms such as acute sepsis, chronic necrosis enteritis, intractable diarrheas; Often cause the large quantities of morbidities of weanling pig; As occur together or other disease of secondary infection or treat untimelyly, mortality ratio is higher, causes heavy losses.Animal infects Salmonellas before death or its product is polluted, and the people is poisoned by food, so this cause of disease is also significant on public hygienics.
People prevent salmonellal typhoid fever with the whole-bacterial-vaccine of deactivation the earliest; Because deactivation vaccine has shortcomings such as causing whole body and local reaction easily, can only excite humoral immunization, cross protection can not be provided, need repeatedly booster immunization, the application of this type of vaccine is restricted.Subsequently, it is found that a little less than malicious Salmonellas induce aspect body fluid and the cell immune response than deactivation or subunit vaccine more effective.So malicious seedling has worldwide obtained widespread use a little less than the various Salmonellass, obtained the good preventing effect.China is at the beginning of the sixties, and the Salmonella choleraesuls virulent strain that Fang Xiaowen etc. are good with antigenicity is selected the good low virulent strain of a strain immunogenicity, called after C500 after inoculating and passing hundreds of generations in the substratum that contains thaliium acetate.C500 promotes the use of in the whole nation, makes China's necrotic enteritis be effectively controlled (Huang Chang Ping, Feng Wenda, Xue Minquan; Zhang Xuji, Yang Shaozhang, Yang Shaojun, Dou Yinmei; Jia Shungeng. malicious aerobic culture freeze-dried vaccine oral immunity research a little less than the necrotic enteritis. Scientia Agricultura Sinica, 1981,6:89 ~ 94; Kang Kai. living paratyphoid vaccine for piglets. Chinese veterinary drug magazine, 2003,37:49).
Over nearly 30 years, people begin pay close attention to use attenuation salmonella as the various exogenous antigens of vector expression to prevent great this research field of people, animal transmissible disease.Using the range gene engineering method makes up the Salmonellas attenuated strain and utilizes attenuation salmonella to express exogenous antigen development polyvalent vaccine etc. as live vector to have become this hot research fields.Attenuation salmonella has many advantages as vaccine carrier: what (1) produced cytotoxic T cell kills and wounds reaction (ctl response); (2) attenuation salmonella can more effectively excite host's body fluid and cellular immunization because of invading lymphsystem; (3) through natural infection approach such as oral or intranasal vaccination, can activating system and mucosal immunoreaction; (4) has immunoadjuvant function; (5) immunity has persistence; (6) have the combined immunization effect, can stably express protokaryon and eukaryotic gene, itself is not pathogenic, and as antityphoid vaccine; (7) expressed target antigen need not purified, and preparation simply can directly be used for the immunoprotection test, has significantly reduced the cost of manufacture of vaccine; (8) inoculation is simple and convenient, (Curtiss, R. such as easy handling; III, T.Doggett, A.Nayak; And J.Srinivasan.1996.Strategies for the use of live recombinant avirulent bacterial vaccines formucosal immunization, and M.F.Kagnoff (ed.) p.499-511.InH.Kiyono, Essentials of mucosalimmunology.Academic Press; San Diego, Calif; S.Spreng, G.Dietrich and G.Weidinger.2006.Rational design of Salmonella-based vaccination strategies.Methods 38:133-143; Sirard, J.C., F.Niedergang, and J.P.Kraehenbuhl.1999.Live attenuated Salmonella:a paradigm of mucosalvaccines.Immunol.Rev.171:5-26).The development of recombinant salmonella vaccine usually need be used selective marker owing to generally adopted in the past the expression plasmid that carries resistant gene, resistant gene become vitro conversion screening transformant and plasmid external heredity main mark.But microbiotic is the main medicine of treating infectation of bacteria clinically, and the maximum fear that causes thus is that resistant gene is propagated in the 20th-century disease pathogenic microorganism, thereby weakens the effect of drugs of these pathogen infections of treatment.Therefore, antibiotics resistance plasmid selective system is not accepted by people.Nowadays; People have developed the multiple salmonella vaccine system that does not contain the antibiotics resistance mark; As with the exogenous antigen stable integration to Salmonellas karyomit(e), or with screening recombinate bacterium but do not need antibiotic plasmid equilibrium system (Balanced-lethal Plasmid Stabilisation System) of selection.Wherein, using maximum systems is asd plasmid vector balanced lethal system.The asd genes encoding aspartic acid beta galactose desaturase (aspartate L-semialdehyde dehydrogenase) of Salmonellas; Be diaminopimelic acid (diaminopimelic acid; DAP) indispensable enzyme in the biosynthetic pathway; And DAP is a component of gram-negative bacteria cell wall main chemical compositions Polysaccharides, peptide complexes tetrapeptide side chain, and the strain of asd disappearance can not form intact cell walls under no external source DAP condition, and final bacteriolyze is dead.Owing to do not contain DAP in the mammalian tissues, so mutant bacteria is degraded all in the substratum that does not contain DAP or in the animal body.Goal gene is inserted the plasmid that contains asd, can form complementation behind the conversion asd host bacterium, the asd Salmonellas of asd plasmid loss is dead in vivo, and the Salmonellas that only contains the asd plasmid could survive, and the stable exogenous antigen of vivoexpression in vivo.Since replaced the antibiotics resistance mark with asd plasmid vector balanced lethal system, therefore also safer.(Nakayama，K.，S.M.Kelly，and?R.Curtiss?III.1988.Coe?N?E?and?R?L?Wood.The?effect?of?exposure?to?aΔcrpΔcya?mutant?of?Salmonella?typhimurium?on?the?subsequent?colonization?of?swine?by?thewild-type?parent?strain.Vet?Microbiol，1992，31：207-220.Hassan，J?O，and?R?Curtiss?III.Controlof?colonization?by?virulent?Salmonella?typhimurium?by?oral?immunization?of?chickens?withavirulentΔcrpΔcya?S.typhimurium.Res.Microbiol，1990，141：839-850.Kelly?S?M，BoseckerB?A.Curtiss?R?III.Characterization?and?protective?properties?of?attenuated?mutants?of?Salmonellacholeraesuis.Infect?Immun，1992，60：4881-4890Construction?of?an?Asd+expression-cloning?vector：stable?maintenance?and?high?level?expression?of?cloned?genes?in?a?Salmonella?vaccine?strain.Bio/Technology?6：693-697；Kang，H.Y.，J.Srinivasan，and?R.Curtiss?III.2002.Immune?responsesto?recombinant?pneumococcal?PspA?antigen?delivered?by?live?attenuated?Salmonella?enterica?serovarTyphimurium?vaccine.Infect.Immun.70：1739-1749.)。
Transmissible gastroenteritis of swine (Transmissible gastroenteritis of pigs; TGE) be by transmissible gastro-enteritis virus (Transmissible gastroenteritis virus; TGEV) a kind of of the pig that causes is the height contagious disease (Leman et al, 1992) of principal character with vomiting, watery diarrhea.TGEV mainly is present in jejunum and the duodenum of pig, is ileum secondly.The equal easy infection of each pig in age, owing to do not set up stable microecosystem as yet in the intestine of young pigs, self resistibility is lower; Stimulate responsive to external world; Be subject to the invasion and attack of various pathogenic micro-organisms and the influence of various stressors, therefore, often because of the death of dewatering; Especially the highest with piglet sickness rate, mortality ratio in 10 ages in days, mortality ratio is up to 100%.5 ages in week, above piglet mortality ratio was lower, and adult pig does not almost have death, but usually caused production performance to descend, and price of deed rate reduces, financial loss big (Sirinarumitr et al, 1998; Jones et al, 1997).Although TGE exists already; But people are the processes that progressively develops to its understanding, and this disease took place in 1945 in Doyle and Hutchings (1946) the reported first U.S., betide Japan (Sasahara et al in 1956; 1958), nineteen fifty-seven occurs in Britain (Goodwin and Jennings; 1958), from that time, most countries all has this pathogenetic report in the world.
The TGEV genome is sectional sub-thread positive chain RNA not, and tool is infectious, and molecular-weight average is 6.8 * 10
6Ku, the about 28.6kb of genome total length (type strain is 28.586kb).The expression of gene structure, reproduction process and viral protein all similar (Laude et al, 1990,1993 with other coronavirus; Enjuanes et al, 1995), 3 ' end has Poly (A) tail, and 5 ' end cap minor structure links to each other with a short homing sequence, and the structural order of whole genome is 5 '-ORF1a-ORF1b-S-ORF3a-ORF3b-sM-M-N-7-3 ' (Sune et al, 1990; Cavanaghet al, 1994).
Complete TGEV comprises four kinds of structural protein: a big surface glycoprotein (Spike or S); A little albumen (sM), a complete membrane glycoprotein (M), and a nucleocapsid protein (N) (Garwes et al, 1979; Godet et al, 1992), respectively the form that virus gets into host cell, virus particle take place and the dispose procedure of virus in play a role, different strains is different than being listed in.
S gp forms typical petal-shaped cyst membrane projection, and long 20nm protrudes in the virus particle surface, and whole virus particle is observed as the imperial crown shape under Electronic Speculum.The antigenic structure of S gp can be divided into antigen site (sites), antigen sublocus (subsities) and antigenic determinant (epitoes).Monoclonal antibody is demarcated and is shown that antigen site divides A, B, C, D totally 4 (Gebauer et al; 1991); (Correa et al such as Correa; 1990) 4 antigen sites of research proof all are positioned at S gp N-and hold 543 amino acid regions of about 1/2, are only accounted for the gene fragment coding (Delmas et al, 1990) of 2.2kb by S gene 5 ' end.The A of different isolates, B and D site high conservative, then there are some variations in the C site.Experiment in vitro proves, A, D site play an important role inducing to produce in the neutralizing antibody, and amino acid guards, and also possibly in the breeding of virus, work.As if the sequence deletion in coding A, D site, its expression product then can not produce neutralizing antibody in the S gene.And other sites (B, C) also can induce neutralizing antibody (Simkins et al, 1992,1993; Correa et al, 1990; Delmaset al, 1990; Gebauer et al, 1991).Variant with anti-TGEV monoclonal antibody confirms that the A site in four major antigen sites can be divided into Aa, Ab, an Ac3 sublocus (Correa et al; 1990); S albumen has 11 antigenic determinants, and wherein 5 is the important antigenic determinant relevant with neutralizing effect.Using the corresponding amino-acid residue of antigenic determinant scanning location sublocus confirms; Amino-acid residue 538,591 and 543 plays keying action respectively for the formation to the Aa in A site, Ab, an Ac3 sublocus; And 586 of residues influence the formation of Aa, Ab sublocus simultaneously, and these presentation of results A site has complicated spatial structure.The 537-MKRSGYGQPIA-547 peptide chain is the major portion of sublocus Ac, and it is conservative at 3 coronavirus crowd camber, and is therefore significant aspect preparation virus diagnose reagent and subunit vaccine.The D site exists and 378-395 residue district; The 385th glycocoll (Gly) residue constitutes important epitope in the D site; Glycosylation is little to the antigenicity influence in D site, and is the same with the A site, and the D site also is the antigen site of mainly inducing neutralizing antibody to produce.
N albumen is a kind of acidic protein of phosphorylation, is present in the inside of virus particle, contains 382 amino-acid residues, and its molecular mass is 47ku, exists with the form of ribonucleoprotein complex.The geneome RNA of N albumen and TGEV is closely linked, and forms a spirrillum ribonucleoprotein mixture, forms the nucleocapsid of virus, is the basis that nucleocapsid forms helix structure.This structure and albumen M form a spherical subviral internal core (Risco et al, 1996).N albumen has 3 main T cell antigen sites, is respectively N48 (46-60 residue district), N272 (272-286 residue district) and N321 (321-335 residue district).Wherein N321T cell antigen site can intensive t cell responses, and can with different proteic B cell epitopes among the TGEV collaborative play a role (Anton, 1995).
People find this disease immune control technology that just begins one's study from nineteen forty-six, roughly experience the strong poison of artificial challenge, deactivation vaccine, attenuated live vaccines and recombinant vaccine four-stage [3].Current; Available commercialization vaccine is that the virus with deactivation or attenuation is the basis; These vaccines can produce neutralizing antibody in the immune animal peripheral blood; Bring out secretory IgA production of antibodies and cell-mediated immunity at small intestine, but can not and effective protection (Brim et al., 1994 be provided this; Park et al., 1998; Saif and Wesley, 1999; Changming Liuet al., 2001).Along with development of molecular biology; People explore with recombinant vaccine and prevent the research of TGE more and more; Principle is to be the main immunogens albumen (great majority are S albumen) of vector expression TGEV through recombinant protein and with virus, bacterium or plant; Thereby produce immune antibody (David et al., 1991 in animal body; Godet et al., 1991; Pulford and Britton, 1991; Torreset al., 1996; Tuboly et al., 2000).Yet up to the present these strategies still fail to realize to be not produce sufficient protective immunity (C.Liu et al., 2001) because expressed recombinant protein can be induced in the pig body.Therefore, safer, efficient, the cheap new generation vaccine of exploitation is pressing for of world's pig industry.
When natural infection of recombinant salmonella living vaccine or experimental infection induce anti-any allos serotype Salmonellas to protect; Salmonellas is because of invading lymphsystem, can more effectively excite the host to produce to advantages such as the body fluid of heterologous antigen and cell immune responses.Like this, perhaps the reorganization attenuated live vaccines is to solve the insufficient a kind of feasible method of current transmissible gastro-enteritis virus commercialized vaccine.There are a lot of defectives in the traditional method that makes up the Salmonellas recombinant bacterial strain.As generally adopting the expression plasmid that carries resistant gene in the past, do not accepted by people because of there being the Biosafety problem.Asd plasmid vector balanced lethal system has the expression amount height, advantages such as purifying antibiotic-free resistance marker are stablized, do not needed to exogenous gene expression; In view of the good immunogenicity of Salmonella choleraesuls C500, it is developed as the live recombined vaccines of expressing transmissible gastro-enteritis virus major antigen locus gene has broad application prospects simultaneously.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists; Its first purpose is the recombinant salmonella choleraesuis strain of expressing transmissible gastro-enteritis virus major antigen site, and this reorganization bacterium contains A, D antigen site and the N of two main generation neutralizing antibodies in the pig infectious gastroenteritis virus S protein of Linker sequence connection of two flexibilities
321The polypeptide of antigen site.
Second purpose of the present invention is to utilize above-mentioned reorganization bacterium to obtain a kind of salmonella choleraesuis strain to prepare Salmonella choleraesuls and transmissible gastro-enteritis virus bivalent genetic engineering vaccine;
The 3rd purpose of the present invention is the application of above-mentioned reorganization bacterium in preparation Salmonella choleraesuls and transmissible gastro-enteritis virus bivalent genetic engineering vaccine.
The present invention realizes through following technical scheme:
The applicant is through engineered method; Obtain a kind of Salmonella choleraesuls (Salmonella choloraesuis) C500/pYA-2SLN of reorganization of the expression transmissible gastro-enteritis virus major antigen site that does not contain resistance marker; This bacterial strain is delivered Chinese typical culture collection center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University on August 31st, 2009, and its deposit number is CCTCC NO:M209189.
The preparation method of a kind of recombinant salmonella choleraesuis C500/pYA-2SLN, it comprises the following steps:
1) synthetic transmissible gastro-enteritis virus major antigen locus gene fragment SLN: the Linker sequence through two flexibilities is the A of two of transmissible gastro-enteritis virus (TGEV) main generation neutralizing antibodies, D antigen site and N321 antigen site in order: D antigen site → Linker → N321 antigen site → Linker → A antigen site is linked in sequence; Synthetic transmissible gastro-enteritis virus major antigen locus gene fragment SLN, its nucleotide sequence is shown in sequence table SEQ ID NO:1;
2) construction recombination plasmid pET-2SLN: the SLN sequence subclone after will synthesizing adopts SalI and XhOI that plasmid pET-SLN is carried out enzyme and cuts and be connected to the pET-28a carrier, and structure obtains the SLN fragment of 2 copies;
3) construction recombination plasmid pYA-2SLN: after the SLN gene fragment of 2 copies that will build is cut connection plasmid pYA3493 from recombinant plasmid pET-2SLN enzyme; Electricity is converted into the strain of Salmonella choleraesuls C500 Δ crp Δ asd disappearance, and obtaining deposit number is the recombinant salmonella choleraesuis C500/pYA-2SLN of CCTCC NO:M209189.
The applicant obtains the gene fragment SLN (nucleotide sequence) in a kind of synthetic transmissible gastro-enteritis virus major antigen site, it is characterized in that, its nucleotide sequence is shown in sequence table SEQ ID NO:1 (its preparation method is seen shown in the embodiment).
The applicant utilizes described recombinant salmonella choleraesuis C500/pYA-2SLN bacterium liquid and gelatin to prepare successfully a kind of bivalent genetic engineering vaccine of preventing and treating Salmonella choleraesuls and transmissible gastroenteritis of swine.
The recombinant salmonella choleraesuis strain in the above-mentioned expression transmissible gastro-enteritis virus major antigen site that does not contain resistance marker lacked cell walls essential in the salmonella strain genome form genes involved asd gene (plasmid that need contain the asd gene be present in the thalline or external source add on the substratum of DAP could normal growth, breeding); This recombinant bacterial strain contains exogenous plasmid pYA-2SLN (containing the asd gene) simultaneously, and this recombinant bacterial strain has kept the immunological characteristic of parent strain C500 as the Salmonella choleraesuls attenuated vaccine strain.Plasmid pYA-2SLN can express the N321 antigen site gene (called after SLN of the present invention) in A, D antigen site and the N albumen in asd gene and the pig infectious gastroenteritis virus S protein in this bacterial strain.Wherein, the asd gene expression product provides salmonella strain essential cell walls forms relevant composition.N321 antigen site gene expression product in A, D antigen site and the N albumen provides the good immunogenicity to transmissible gastro-enteritis virus.
The recombinant salmonella choleraesuis strain C500/pYA-2SLN that does not contain the expression transmissible gastro-enteritis virus major antigen site of resistance marker of the present invention, this engineering strain C500/pYA-2SLN are derived from China and have used the commercialization Salmonella choleraesuls attenuated vaccine strain C500 more than 50 years.Described recombinant salmonella choleraesuis strain C500/pYA-2SLN of the present invention; Lack the asd gene in the C500 strain gene group, added the plasmid pYA-2SLN of the N321 antigen site gene in A, D antigen site and the N albumen that contains in the pig infectious gastroenteritis virus S protein simultaneously.Owing to need the recombinant bacterial strain that exists of pYA-2SLN to survive, therefore improved the stability of plasmid pYA-2SLN (just A, D antigen site and N321 antigen site gene) in recombinant bacterial strain greatly.A, D antigen site and the stably express of N321 antigen site gene in recombinant bacterial strain make it to have to the good immune protective efficiency of transmissible gastro-enteritis virus.
Basic construction method of the present invention is: the Salmonella choleraesuls attenuated vaccine strain C500 Δ crp Δ asd disappearance strain of the asd genetically deficient that the agriculture mikrobe National Key Laboratory that utilizes that the applicant belongs to builds is (referring to document: Xu Yindi etc.; The structure and the evaluation of Salmonella choleraesuls C500 strain Δ crp Δ asd disappearance strain balanced lethal carrier system. the biotechnology journal; 2006; 5 (3): 366-371), make up the plasmid pYA-2SLN that contains the N321 antigen site gene in A, D antigen site and the N albumen in the pig infectious gastroenteritis virus S protein (GenBank No:DQ443743) simultaneously.Plasmid pYA-2SLN is changed in the C500asd gene-deleted strain; Owing to growth need that plasmid pYA-2SLN and C500asd gene-deleted strain are complementary stablizes coexistence, form recombinant C 500 (pYA-2SLN) bacterial strain in the expression transmissible gastro-enteritis virus major antigen site that does not contain resistance marker that we invent.Recombinant bacterial strain through a large amount of biological experiment digital proof the present invention preparations can be used for preparing the bivalent genetic engineering vaccine to Salmonella choleraesuls and transmissible gastro-enteritis virus.
Major advantage of the present invention is:
1, the N321 antigen site in A, D antigen site and the N albumen in the pig infectious gastroenteritis virus S protein of recombinant bacterial strain expression of the present invention is the important immunogenic gene fragment of transmissible gastro-enteritis virus, has good immune protection property.Because transmissible gastro-enteritis virus harm is serious day by day, and extensive stock transmissible gastroenteritis of swine vaccine (mainly being adjuvant inactivated vaccine) immune effect is generally relatively poor in the market.Therefore, the vaccine of processing with this engineering strain has wide market application prospect.
2, recombinant bacterial strain of the present invention is derived from China and has used the commercialization Salmonella choleraesuls attenuated vaccine strain C500 more than 50 years, has kept the immune efficacy of C500 to Salmonella choleraesuls fully.And, this recombinant bacterial strain virulence than C500 slightly a little less than, have better biological safety.
3, recombinant bacterial strain of the present invention can provide the protection to Salmonella choleraesuls and two kinds of cause of diseases of TGE simultaneously.
4, recombinant bacterial strain of the present invention does not contain resistance marker, meets the requirement of vaccine biological safety fully.
More detailed technical scheme sees that embodiment is said.
Description of drawings
Sequence table SEQ ID NO:1 is the gene fragment SLN (single copy) in synthetic transmissible gastro-enteritis virus major antigen of the present invention site.
Fig. 1: the physical map that is the recombinant plasmid pYA-2SLN for preparing of the present invention.
Fig. 2: the PCR that is the recombinant plasmid pYA-2SLN for preparing of the present invention identifies electrophoretogram.
M:DNA Marker (DL2000) 1:pYA-2SLN 2:pYA3493 among the figure
Fig. 3: the enzyme that is the recombinant plasmid pYA-2SLN for preparing of the present invention is cut qualification result.
M1:DNA Marker (DL2000) among the figure, M2:DNA marker (DL 15,000) 1:pYA-2SLN/EcoR I+Hind III
2：pYA3493/EcoR?I+Hind?III
Fig. 4: the genetic stability experimental result that is a kind of recombinant salmonella choleraesuis living vaccine bacterial strain of the expression transmissible gastro-enteritis virus major antigen site that does not contain resistance marker among the present invention.M:DNA marker among the figure (DL 2,000); 7 is C500 (pYA3493) contrast; 1-6 is that 1-50 is for recombinant bacterial strain
Fig. 5: be the schema that the transferring plasmid pYA-SLN for preparing of the present invention makes up.
Fig. 6: the SDS-PAGE and the Western-blot analytical results that are a kind of recombinant salmonella choleraesuis living vaccine bacterial strain C500 (pYA-2SLN) of the expression transmissible gastro-enteritis virus major antigen site that does not contain resistance marker among the present invention.
Fig. 6 A detects fusion rotein Cpro-2SLN with the SDS-PAGE method, and 1 is C500 (pYA3493) among the figure; 2 is recombinant bacterial strain C500 (pYA-2SLN); Arrow is depicted as amalgamation and expression PROTEIN C pro-2SLN.
Fig. 6 B detects fusion rotein Cpro-2SLN with the Western-blot method, and 1 is C500 (pYA3493) among the figure; 2 is recombinant bacterial strain C500 (pYA-2SLN)
Fig. 7: the collection of illustrative plates that is recombinant salmonella used carrier among the present invention.
Fig. 8: be 3 antigen site sequences in the transmissible gastro-enteritis virus (TGEV).
Fig. 9: be splicing, synthetic transmissible gastro-enteritis virus (TGEV) major antigen site sequence, among the figure: thickened portion is the Linker sequence, and the character frame is a restriction enzyme site, and underscore partly is the point mutation site.
Embodiment
Below in conjunction with Figure of description the present invention is further described.
It is segmental synthetic that embodiment 1 contains transmissible gastro-enteritis virus major antigen locus gene
1, the selection in major antigen site
Pig infectious gastroenteritis virus S protein contains A, B, C, D totally 4 antigen sites, and wherein A and D antigen site play an important role inducing to produce in the neutralizing antibody, and the A antigen site is a main B cell antigen epi-position.Transmissible gastro-enteritis virus contains 4 main t cell epitopes, wherein the N on the N albumen
321(321-335 residue district) cell antigen site can intensive t cell responses, and can play a role with different proteic B cell epitopes among the TGEV are collaborative.
2, the design of S and N gene primer and the comparison of homology thereof
With reference to the pig infectious gastroenteritis virus S of having reported and 2 pairs of primers of N (GenBank No:DQ443743) gene order design, from the pathological material of disease that contains transmissible gastro-enteritis virus, amplify S gene and N gene respectively through the RT-PCR method.Amplification purpose clip size is respectively 2331bp and 1440bp, and pS1 and pS2 introduce EcoR I and Sal I restriction enzyme site respectively, and pN1 and pN2 introduce EcoR I and Sal I restriction enzyme site respectively.Primer sequence is following:
pS?1：5’-GGG
GAATTCATGAAAAAACTATTTGTGG-3’(EcoRI)
PS2:5 '-ACT
GTCGACGGCTTGTGCGCTTAC-3 ' is S fragment 2331bp (SalI)
pN1：5’-CAT
GAATTCATGGCCAACCAGG-3’(SalI)
PN2:5 '-GCC
GTCGACGTTAGTTCGTTACC-3 (Hind III) N fragment 1440bp
Through RT-PCR amplify be connected the T-carrier behind S and the N gene after, check order.Sequencing result shows; The S of the TGEV that preserve this chamber and N gene and 8 strain TGEV both domestic and external (Purdue (U.S.), NC-002306 (Spain), Purdue P115 (U.S.); MillerM60 (U.S.); MillerM6 (U.S.), TS (Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences), the homology of SC-Y (Sichuan Agricultural University) and TH-98 (Northeast Agricultural University) is between 97%-99%.Wherein at A, D and N
321On the antigen site, with the domestic SC-Y and the homology of TH-98 strain be 100%.
3, the design of SLN sequence is with synthetic
Linker sequence through two flexibilities couples together the A of two of TGEV main generation neutralizing antibodies, D antigen site and N321 antigen site (order is D antigen site sequence → Linker → N321 antigen site → Linker → A antigen site sequence), and this sequence called after SLN.Carry out point mutation (GTG → GTa), but do not change its amino acids coding to detesting codon.(AAC → Aat, ATC → Att, ACA → ACt) carry out point mutation, but do not change its amino acids coding to 3 sites that methylate on the A antigen site.Introduce SalI restriction enzyme site and NotI restriction enzyme site respectively at the upstream and downstream of SLN.Concrete sequence is seen Fig. 9.
The SLN sequence that designs is sent to Shanghai invitrogen company synthesizes.
The structure of embodiment 2 recombinant plasmid pYA-2SLN
1, the clone of SLN gene
(DH5 α/pET-2SLN) is to be transferred in the bacterium bottle that contain 3mL LB substratum 37 ℃, 200rpm/min incubated overnight 8h at 1: 100 according to volume ratio will to contain the reorganization bacterium of SLN gene order.Extracting test kit (available from Beijing BioFlux company) specification sheets extraction plasmid by bacterial plasmid is that enzyme is cut template.
With EcoRI and NotI double digestion SLN and pET-28a carrier (available from precious biotechnology (Dalian) ltd); Connect with T4DNA ligase after reclaiming purifying, 16 ℃ of water-bath 12h transform DH5 α competence bacterium; Cultivate 12h for 37 ℃; Choose bacterium to liquid LB substratum, 37 ℃, 200rpm/min jolting cultivation 12h obtain plasmid pET-SLN (see figure 5) thereby prepare plasmid in a small amount.
2, the structure and the design of primers of series connection multiple copied SLN gene
Utilizing last SalI of pET-SLN and XhOI is the character of one group of isocaudarner, and part pET-SLN plasmid is carried out SalI and SmaI double digestion, and another part pET-SLN plasmid carries out XhOI and SmaI double digestion.All produce two fragments behind every group of plasmid double digestion, will contain the SLN gene order, promptly select small segment in SalI and the SmaI double digestion group, select large stretch of degree to reclaim purifying in XhOI and the SmaI double digestion group.The SalI enzyme is cut the back and is produced 3 '-GCTG, and the sticky end that produces after can cutting with the XhOI enzyme matches.Through connecting, can construct the recombinant plasmid that contains 2 SLN gene orders, this moment, the SalI and the XhOI restriction enzyme site of junction disappeared, and had guaranteed that pET-2SLN goes up the singularity (see figure 5) of SalI and XhOI restriction enzyme site.
Can construct the recombinant plasmid that contains 3 or 4 copy SLN genes on this basis.
The primer sequence that is used to detect pET-2SLN is following:
PSLNI (upstream primer): 5 '-ACTGTCGACCTTCTGTAAGTGATTCGAGC-3 '
PSLNII (downstream primer): 5 '-TTAAGCGGCCGCTCGTGCAG-3 '
3, the structure of recombinant plasmid pYA-2SLN and evaluation
With EcoRI and XhOI double digestion pET-2SLN plasmid, (be so kind as to give by Dr.Roy Curtiss professor III of Washington, DC university with EcoRI and Sal I shuttle plasmid pYA3493; Kang; H.Y.; J.Srinivasan; And R.Curtiss III.2002.Immune responsesto recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovarTyphimurium vaccine.Infect.Immun.70:1739-1749), use T behind the recovery purifying
4DNA ligase connects, 16 ℃ of water-bath 12h, and electric transformed into escherichia coli x 6097 (is so kind as to give by Dr.Roy Curtiss professor III of Washington, DC university; Nakayama; K.; S.M.Kelly, and R.Curtiss III.1988.Construction of an Asd+expression-cloning vector:stablemaintenance and high level expression of cloned genes in a Salmonella vaccine strain.Bio/Technology 6:693-697) competent cell, cultivate 12h for 37 ℃; Choose bacterium to liquid LB substratum; 37 ℃, 200rpm/min jolting cultivation 12h obtain plasmid pYA-2SLN thereby prepare plasmid in a small amount, and its physical map is seen Fig. 5.Qualification result confirms that the recombinant plasmid pYA-2SLN that makes up is correct (see figure 6), and it is as shown in Figure 7 that recombinant plasmid pYA-2SLN makes up flow process.Use T after reclaiming purifying
4DNA ligase connects, and 16 ℃ of water-bath 12h will connect product and be converted into bacillus coli DH 5
αCompetent cell picking list bacterium colony after cultivating 12h under 37 ℃ in 37 ℃, 200rpm/min jolting cultivation 12h, obtains plasmid pYA-2SLN thereby prepare plasmid in a small amount to the LB liquid nutrient medium, its physical map is seen Fig. 5.Qualification result confirms that the transferring plasmid pYA-2SLN that present embodiment makes up is correct (see figure 6), and it is as shown in Figure 7 that transferring plasmid pYA-2SLN makes up flow process.
The primer sequence that is used to detect pYA-2SLN is following:
pYASLNI：5’-GCTGAAGAATTCGAGCTCCGTCGACCTTC-3’
pYASLNII：5’-CTTGGGAGCTTGGCTGCAGGTCGAG-3’
Recombinant shuttle plasmid pYA-2SLN (embodiment 2 is seen in the source) electricity is transformed (parameter: voltage 2.0KV; Time 4ms; 200 ohm of electric capacity 25 μ F and pulse resistances) to the strain of Salmonella choleraesuls C500 Δ crp Δ asd disappearance, at the negative dull and stereotyped enterprising row filter positive colony of DAP, picking list bacterium colony is cultivated; And carry out PCR with primer pYASLNI/pYASLNII and identify (PCR reaction system (25 μ L): 10 * Buffer, 2.5 μ L, 25mmol/L MgCl
22 μ L, 2 μ mol/L dNTPs, 1 μ L, each 1 μ L of 10 μ g/mL upstream and downstream primers, 2U/ μ L Taq archaeal dna polymerase 0.5 μ L, sterilized water 16 μ L, template 1 μ L.PCR reaction conditions: 94 ℃ of sex change 4min; Get into 25 circulations then, 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 60s; Last 72 ℃ are extended 10min.The PCR product is the 80V electrophoretic separation on 1.0% sepharose, ethidium bromide (EB) dyeing, gel imaging system scanography), its result sees respectively shown in Fig. 8 A, Fig. 8 B and Fig. 8 C.The result shows that the recombinant bacterial strain of the Salmonellas C500 that obtains to contain the pYA-2SLN plasmid is correct, and the applicant is with its called after salmonella choleraesuis strain (Salmonellacholeraesuis) C500/pYA-2SLN and deliver the preservation (preservation information sees that " summary of the invention " is said) that Chinese typical culture collection center (CCTCC) is used for patented procedure.
1, the preparation of SLN gene fragment e. coli expression product mouse-anti serum
The pET28a-2SLN plasmid is carried out sequencing; After confirming that the clone is correct, transformed into escherichia coli BL21 (DE3) is containing kantlex (Kan; Final concentration is 50 μ g/mL) on the flat board picking positive transformant to liquid LB substratum; 37 ℃, 200rpm/min are cultured to logarithmic phase (OD=0.6-1.0) and add IPTG (final concentration 1mM) and carry out abduction delivering 4h, by producer's process specifications, use the Histidine fusion rotein to extract test kit HIS-Bind Purification Kit (this test kit is available from U.S. Qiagen company) the pET28a-2SLN expression product is carried out purifying; Obtaining concentration is the amalgamation and expression albumen of 230 μ g/mL, called after 2SLN-Pro.With 100 μ g expression product 2SLN-Pro and equal-volume Freund's complete adjuvant uniform mixing, subcutaneous injection BALB/C mice, each booster immunization 1 time (use Freund's incomplete adjuvant) after 3 weeks (two exempt from) and 5 weeks (three exempt from) respectively.Exempt from back 10d blood sampling 3, extract serum, 0.22 μ m membrane filtration degerming, put-20 ℃ subsequent use.
2, the expression characterization analysis of C500 (pYA-2SLN) recombinant bacterial strain
Single bacterium colony of picking recombinant bacterial strain C500/pYA-2SLN is in the LB liquid nutrient medium, and 37 ℃, 200rpm/min are cultivated 16h, 8; The centrifugal collection thalline of 000r/min, carry out the polyacrylamide gel electrophoresis (SDS-PAGE, method is with reference to [U.S.As] such as J. Sa nurse Brookers; " molecular cloning experiment guide ", Science Press, 2003; And use mouse-anti HIS-SLN serum to carry out Western-blotting and analyze that (method is with reference to [U.S.As] such as J. Sa nurse Brookers, " molecular cloning experiment guide " third edition); Science Press, 2003, the third edition).The result shows that the C500/pYA-2SLN that the present invention prepares can express the recombinant protein (called after Cpro-2SLN) that size is respectively 37.67kDa, and the recombinant protein of expressing has good immunological response originality (see figure 9).
3, the phenotypic evaluation of recombinant bacterial strain C500/pYA-2SLN
Recombinant bacterial strain C500/pYA-2SLN and parent plant C500 streak inoculation is dull and stereotyped in LB, and carbon source and H such as SANMALT-S, glucose, lactose, sucrose, rhamnosyl, seminose, pectinose then transfer
2Biochemical identification pipes such as S carry out biochemical reaction.The result shows that the biochemical characteristic of recombinant bacterial strain C500 (pYA-2SLN) is consistent with parent plant C500.Both all can not produce H
2S can not utilize lactose, sucrose, pectinose and urea, but can utilize SANMALT-S, glucose, rhamnosyl and seminose is unique charcoal source.The result shows that the biochemical characteristic of recombinant bacterial strain C500 (pYA-2SLN) is consistent with parent strain C500, meets the typical phenotypic characteristic of Salmonella choleraesuls.
4, the growth characteristics analysis of recombinant bacterial strain C500/pYA-2SLN
Recombinant bacterial strain C500/pYA-2SLN is cultivated 12h for dull and stereotyped 37 ℃ at LB, and its diameter of bacterium colony is about 1.8mm, and is slightly littler than C500 strain (2.1mm) than the parent.Parent plant C500 and C500/pYA-2SLN recombinant bacterial strain are from 10
6CFU/ml begins to cultivate, every 1h sampling, and carry out live bacterial count.The result shows that the recombinant bacterial strain C500/pYA-2SLN speed of growth is slower than parent plant C500 slightly, and its average length of generation (Mean generation time) is 30.3min, prolongs 2.4min than parent plant (27.9min).
5, the genetic stability of recombinant bacterial strain C500/pYA-2SLN
The C500/pYA-2SLN recombinant bacterial strain of the present invention preparation is streak culture on the LB solid plate, picking list bacterium colony in the LB liquid nutrient medium, 37 ℃; 200r/min cultivates 16h; 1: 1 by volume, 000 ratio was transferred in the LB liquid nutrient medium and cultivates 12h, once more by 1: 1; 000 ratio is transferred in the LB liquid nutrient medium, carries out 10 switchings continuously.Carry out pcr amplification with primer pSLNI/pSLNII, the hereditary situation of amplification plasmid in the reorganization bacterium.(see figure 10) shows as a result, and the result of each amplification does not all have observable difference, shows that the C500/pYA-2SLN recombinant bacterial strain that the present invention prepares can genetic stability.
The C500/pYA-2SLN recombinant bacterial strain that obtains is identified; In per generation, be inoculated on the LB substratum; Utilize primer pSLNI/pSLNII to carry out the genetic stability that PCR detect to identify recombinant bacteria, discovery still can amplify size and be respectively 980bp fragment, genetic stability after going down to posterity for 20 times.Detect to find through Western-blotting, the Cpro-2SLN recombinant protein can be in recombinant bacterial strain C500/pYA-2SLN stably express, and have good immunological response originality.This recombinant bacterial strain C500/pYA-2SLN cultivates on the LB solid medium, and picking list bacterium colony is cultivated in the LB liquid nutrient medium, reaches 2.5 * 10 up to viable bacteria concentration
10CFU/mL.With the centrifugal back of bacterium liquid by bacterium liquid (reorganization bacterium bacterium liquid C500/pYA-2SLN of the present invention): gelatin protective material (volume: be that 2: 1 ratio adds the gelatin protective material (this gelatin protective material compound method is: with sucrose 40g, gelatin 8g is fully after the thawing in every 100mL deionized water volume); Preservation is subsequent use after putting 121 ℃ of 30min that sterilize down); In sterilization freeze-drying bottle, press the packing of 2.0mL/ bottle, put freeze-drying in-50 ℃ of freeze driers, freeze-drying 36-40h rear pressing cover; With physiological saline solution and carry out live bacterial count (CFU); And confirm there is not living contaminants, it is subsequent use to put-20 ℃ of preservations, as the vaccine strains of development recombiant vaccine.
1, the immune programme for children of mouse:
The BALB/c mouse that uses 5-6 age in week is divided into 3 groups as the immune efficacy evaluation according to TR, is respectively recombinant bacterial strain C500/pYA-2SLN vaccine group, C500 parent strain vaccine group and the non-immune blank group of the present invention's preparation.Immunization route is that oral 0.2mL (contains 2.5 * 10
9CFU viable bacteria amount) bacterium liquid or LB substratum, booster immunization is 1 time after 14 days.Exempt to detect in back 14 days and 28 days Salmonellas serum antibody (ELISA respectively at preceding 0 day of immunity, head; Working method is with reference to Xu Yindi. the structure and the application of Salmonella choleraesuls C500 strain crp-, asd-disappearance strain balanced lethal system. [doctorate paper]. Wuhan: Hua Zhong Agriculture University Library, 2006.).Histidine fusion expressed product 2SLN-Pro with preparation among the embodiment 4 is that antigen (1750ng/ hole) encapsulates elisa plate simultaneously; (method is with reference to Liu Zhong brightness and Lv Changlong to press indirect ELISA method; Immunology common experimental technology; Beijing: Science Press, 2002) detect Cpro-2SLN recombinant protein IgG and IgA antibody horizontal.
2, immune mouse humoral immunization antibody horizontal detects
Separate small intestine before the mouse immune, second and third time separation small intestine is exempted from the back at head respectively and was carried out in 14,28 days, chooses 5 for every group and adds PBS through separating small intestine, grinding back centrifuging and taking supernatant.Detect anti-salmonella antibody respectively, Cpro-2SLN specificity IgA antibody is averaged.The result sees table 3.From table 3, can find out; It is 1.30 and 1.28 that head exempts from back the 2nd week OD630 value of recombinating bacterium C500/pYA-2SLN vaccine group and C500 parent strain group antibodies toward salmonella of the present invention; Two exempt from 2 weeks of back (being that head exempts from 4 weeks of back), and the OD630 value of reorganization bacterium C500/pYA-2SLN vaccine group of the present invention and C500 parent strain group antibodies toward salmonella rises to respectively 1.83 and 1.76.The OD630 values that head exempts from 2 week reorganization of the present invention bacterium C500/pYA-2SLN vaccine group CPro-2SLN specific antibodies are 0.81, two to exempt from 2 weeks afterwards, and the OD630 value of reorganization bacterium C500/pYA-2SLN vaccine group Cpro-2SLN antibody of the present invention rises to 1.26.And contrast C500 parent strain group of carrying out synchronously and PBS control group one exempt to be respectively 0.36 to 0.41 and 0.24 to 0.23 with the two antibody OD630 values exempted from.The above results can induce body to produce the HI of specific anti-salmonella and transmissible gastroenteritis of swine after showing the reorganization bacterium C500/pYA-2SLN vaccine immune mouse that the present invention prepares.
The reorganization bacterium C500/pYA-2SLN vaccine immune mouse enteron aisle IgA antibody test (ELISA method) of table 3 the present invention preparation
Since in January, 2009, successively at the recombiant vaccine of A, B, C and about 2.9 ten thousand parts of D pig farm oral immunity.Before A did not use the field this seedling, the piglet case fatality rate was about 60%.Behind the oral bivalent genetic engineering vaccine of the present invention, survival rate is 99%.Behind the piglet oral vaccine of morbidity, diarrhoea waits transference cure in 24-48 hour.Behind other each oral bivalent genetic engineering vaccines of the present invention, also receive the effect similar with the A field.
Sequence table
< 110>Hua Zhong Agriculture University
< 120>recombinant salmonella choleraesuis strain and the application in expression transmissible gastro-enteritis virus major antigen site
<130>
<141>2009-09-10
<160>1
<170>PatentIn?version?3.1
<210>1
<211>441
<212>DNA
< 213>transmissible gastro-enteritis virus
<220>
<221>gene
<222>(1)..(441)
<223>
<400>1
actgtcgacc?ttctgtaagt?gattcgagct?ttttcagtta?cggtgaaatt?ccgttcggcg 60
taactgatgg?accacggtac?tgttacggtg?gcggtggctc?tggcggaggt?gggagcggcg 120
gtggtggcag?ccaattcctt?cagcagatta?atgcctatgc?tcgtccatca?gaagtagcaa 180
aagaacagag?aaaaagaaaa?tctcgtggtg?gtggcggttc?tggcggaggt?ggcagcggcg 240
gtggcggttc?tggtatgaag?cgtagtggtt?atggtcaacc?catagcctca?acattaagta 300
atattactct?accaatgcag?gatcacaaca?ccgatgtata?ctgtattcgt?tctgaccaat 360
tttcagttta?tgttcattct?acttgcaaaa?gtgctttatg?ggacaatatt?tttaagcgaa 420
actgcacgag?cggccgctta?a 441
Claims (5)
1. recombinant salmonella choleraesuis (Salmonella choloraesuis) C500/pYA-2SLN in transmissible gastro-enteritis virus major antigen site is expressed in a strain, is deposited in Chinese typical culture collection center, and its deposit number is CCTCC NO:M209189.
2. the preparation method of a recombinant salmonella choleraesuis C500/pYA-2SLN, it comprises the following steps:
1) synthetic transmissible gastro-enteritis virus major antigen locus gene fragment SLN: the Linker sequence through two flexibilities is the A of two of transmissible gastro-enteritis virus main generation neutralizing antibodies, D antigen site and N321 antigen site in order: D antigen site → Linker → N321 antigen site → Linker → A antigen site is linked in sequence; Synthetic transmissible gastro-enteritis virus major antigen locus gene fragment SLN, its nucleotide sequence is shown in sequence table SEQ ID NO:1;
2) construction recombination plasmid pET-2SLN: the SLN sequence subclone after will synthesizing adopts SalI and XhOI that plasmid pET-SLN is carried out enzyme and cuts and be connected to the pET-28a carrier, and structure obtains the SLN fragment of 2 copies;
3) construction recombination plasmid pYA-2SLN: after the SLN gene fragment of 2 copies that will build is cut connection plasmid pYA3493 from recombinant plasmid pET-2SLN enzyme; Electricity is converted into the strain of Salmonella choleraesuls C500 Δ crp Δ asd disappearance, and obtaining deposit number is the recombinant salmonella choleraesuis C500/pYA-2SLN of CCTCC NO:M209189.
3. the gene fragment SLN in a synthetic transmissible gastro-enteritis virus major antigen site is characterized in that, its nucleotide sequence is shown in sequence table SEQ ID NO:1.
4. recombinant salmonella choleraesuis and transmissible gastroenteritis of swine bivalent genetic engineering vaccine is characterized in that this vaccine contains the recombinant salmonella choleraesuis C500/pYA-2SLN that preserving number is CCTCC NO:M209189.
5. the application of the described recombinant salmonella choleraesuis of claim 1 in preparation Salmonella choleraesuls and transmissible gastroenteritis of swine bivalent genetic engineering vaccine.
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| CN102732472A (en) * | 2011-04-08 | 2012-10-17 | 华中农业大学 | Construction and application of attenuated Salmonella choleraesuis for expressing mycoplasma hyopneumoniae p65 protein |
| CN103013895B (en) * | 2011-09-23 | 2014-07-16 | 华中农业大学 | Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application |
| CN103421731B (en) * | 2013-01-31 | 2015-10-28 | 华中农业大学 | A kind of haemophilus parasuis attenuation salmonella seedling |
| CN103421733B (en) * | 2013-01-31 | 2015-10-28 | 华中农业大学 | A kind of haemophilus parasuis-Salmonella choleraesuls bigeminy gene engineering vaccine |
| CN107158371A (en) * | 2017-05-16 | 2017-09-15 | 青岛农业大学 | A kind of gene engineered subunit bigeminy oral vaccine |
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| Title |
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| 徐引弟等.猪霍乱沙门氏菌C500株△crp△asd缺失株平衡致死载体系统的构建及鉴定.《生物工程学报》.2006,第22卷(第3期),366-372. * |
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