CN101838627A - Recombination salmonella choleraesuis and bivalent genetic engineering vaccine and preparation method - Google Patents

Abstract

The invention belongs to the technical field of the animal bacteria genetic engineering and in particular relates to a structure of recombination salmonella choleraesuis, a bivalent genetic engineering vaccine and a preparation method and application of the recombination salmonella choleraesuis, wherein the recombination salmonella choleraesuis does not comprise a resistance marker and expresses main antigen Cap proteins of porcine circovirus II. The preservation number of the recombination salmonella choleraesuis C501 (pYA-delta 410RF2) which does not comprise the resistance marker, expresses main antigen sites of porcine circovirus II and is prepared by the invention is CCTCC M209314. The recombination salmonella choleraesuis deletes asd genes which is necessary for the growth of the salmonella choleraesuis and comprises plasmid which can express the asd genes and genes of the antigen sites of the Cap proteins of porcine circovirus II in the recombination salmonella choleraesuis. The invention discloses the method and the application for preparing salmonella choleraesuis and the porcine circovirus II bivalent genetic engineering vaccine by utilizing the recombination salmonella choleraesuis. The bivalent genetic engineering vaccine of the invention can stimulate a swine to generate protective immune response resisting the salmonella choleraesuis and the porcine circovirus II and effectively prevent infection of the salmonella choleraesuis and the porcine circovirus II.

Description

Recombinant salmonella choleraesuis and bivalent genetic engineering vaccine and preparation method

Technical field

The invention belongs to the animal bacteria gene engineering technology field, be specifically related to a kind of structure and application that does not contain the proteic recombinant salmonella choleraesuis living vaccine of the expression pig II type PCV-II Cap strain of resistance marker.

Background technology

Salmonella choleraesuls (Salmonella choleraesuis) are the main pathogen that causes 2-4 monthly age necrotic enteritis, can cause classical symptoms such as acute sepsis, chronic necrosis enteritis, intractable diarrhea after the infected pigs, often cause the large quantities of morbidities of weanling pig, as occur together or other disease of secondary infection or treat untimely, 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 weak malicious Salmonellas induce aspect body fluid and the cell immune response than deactivation or subunit vaccine more effective.So the weak malicious seedling of various Salmonellass has worldwide obtained widespread use, has 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.After C500 is promoted the use of in the whole nation, China's necrotic enteritis be effectively controlled (Huang ChangBing etc., the weak malicious aerated culture freeze-dried vaccine oral immunity research of necrotic enteritis. state's agricultural sciences, 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 people, animal transmissible disease research field.Using the range gene engineering method makes up the Salmonellas attenuated strain and utilizes attenuation salmonella to express the research focus that exogenous antigen development polyvalent vaccine etc. has become this field as live vector.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) by 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 can be used as antityphoid vaccine; (7) expressed target antigen need not purified, and preparation is simple, can be directly used in the immunoprotection test, has significantly reduced the cost of manufacture of vaccine; (8) inoculation is simple and convenient, (Curtiss such as easy handling, R., III, T.Doggett, A.Nayak, and J.Srinivasan.1996.Strategies for the use of live recombinant avirulent bacterial vaccines for mucosal immunization, H.Kiyono and M.F.Kagnoff (ed.) p.499-511.In, Essentials of mucosal immunology.Academic Press, San Diego, Calif; S.Spreng, G.Dietrich and G.Weidinger.2006.Rational design of Salmonella-based vaccination strategies.Methods38:133-143; Sirard, J.C., F.Niedergang, and J.P.Kraehenbuhl.1999.Live attenuated Salmonella:a paradigm ofmucosal vaccines.Immunol.Rev.171:5-26).The development of recombinant salmonella vaccine often 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 for the treatment of 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 recombinate bacterium but do not need antibiotic plasmid equilibrium system (Balanced-lethal Plasmid Stabilisation System) with screening.Wherein, using maximum systems is asd plasmid vector balanced lethal system.The asd genes encoding aspartic acid beta galactose desaturase (aspartateL-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 peptidoglycan tetrapeptide side chain, the strain of asd disappearance can not form intact cell walls under no external source DAP condition, final bacteriolyze death.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，andR.Curtiss?III.1988.Coe?N?E?and?R?L?Wood.The?effect?of?exposure?to?a?ΔcrpΔcya?mutant?of?Salmonellatyphimurium?on?the?subsequent?colonization?of?swine?by?the?wild-type?parent?strain.Vet?Microbiol，1992，31：207-220.Hassan，J?O，and?R?Curtiss?III.Control?of?colonization?by?virulent?Salmonella?typhimurium?by?oralimmunization?of?chickens?with?avirulent?ΔcrpΔcya?S.typhimurium.Res.Microbiol，1990，141：839-850.Kelly?SM，Bosecker?B?A，Curtiss?R?III.Characterization?and?protective?properties?of?attenuated?mutants?of?Salmonellacholeraesuis.Infect?Immun，1992，60：4881-4890?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；Kang，H.Y.，J.Srinivasan，and?R.Curtiss?III.2002.Immune?responses?to?recombinant?pneumococcal?PspAantigen?delivered?by?live?attenuated?Salmonella?enterica?serovar?Typhimurium?vaccine.Infect.Immun.70：1739-1749.)。

Pig circular ring virus (Porcine circovirus, PCV) discovery is in 1974, Tischer etc. (1974) are in the process that goes down to posterity of PK-15 clone ATCC-CCL31, found and parvovirus and the similar particle of papovavirus, this particulate material does not cause cytopathy, once is considered to a kind of cell contamination thing at that time.Tischer etc. (1982) extract virus particle and viral nucleic acid with ultracentrifugation, and by electron microscopic observation nucleic acid state, the material that has disclosed this pollution PK-15 clone first is actually a kind of strand ring-type dna virus, and with its called after PCV.

With this virus infection experimental animal, find to have only pig to produce specific antibody, but do not have clinical symptom and pathological change, and rabbit, mouse, Niu Junwei serology feminine gender.A large amount of serosurvey results shows that PCV antibody is positive in the swinery of many countries, and PCV infects very general.Especially 1991 Canada occurred wean pig and growing-finishing pigs slowly, breathe urgent, become thin, anaemia, jaundice, cut open inspection lymphadenectasis, liver cirrhosis, many kitchen ranges property mucus trachitis, interstitial pneumonia and ephritis etc. are arranged, thought a kind of swine disease of New Development at that time, with it called after pmws (Post-weaning pigs multisystemic wasting syndrome, PMWS) (Fenaux et al., 2000).Subsequently, this disease takes place in succession in the whole world rapidly, China also reported in 2000 should disease generation (Lang Hongwu etc., 2000).Studies show that by clinical and breadboard the main pathogen that causes pmws is a pig circular ring virus, but be not unique cause of disease (Allan et al., 1998).Also find the PCV of this cause of disease and traditional persistent infection PK-15 cell simultaneously, antigenicity is similar to each other, but very big difference is arranged also, so otherness that lists at pathogenic, antigenicity and nucleotides sequence according to PCV, the pathogenic PCV that has that will be separated in the sick pig body of PMWS is called PCV2, and the PCV that will pollute PK-15 cell and no pathogenicity be called PCV1 (Allan et al., 1998a).

The PCV genome is a sub-thread ring-type minus-strand dna, and PCV1 genome total length is 1759bp, and PCV2 genome total length is 1767 or 1768bp.The genome homology of two kinds of genotype viruses is 76%, and the PCV internal gene group variation of homogenic type is very little, and genomic homology is greater than 99% between the PCV1 strain isolated, and genomic homology is greater than 96% between the PCV2 strain isolated.The genome of PCV is encoded altogether, and (Openreading frames ORFs), reads between the frame overlappedly 11 open reading frames, makes virus can make full use of the limited a large amount of genetic information of genetic material transmission.ORF1, ORF5, ORF7, the ORF10 of virus are on the viral DNA chain, and ORF2, ORF3, ORF4, ORF6, ORF8, ORF9, ORF11 be on complementary strand, in these 11 open reading frames, and importantly ORF1 and ORF2 on the function.ORF1 coding Rep and Rep ' albumen, with virus genomic duplicate relevant.Nucleocapsid (Cap) albumen of ORF2 coding virus is the unique structural protein of virus.Find by sequential analysis, nucleic acid homology is 83% between the ORF1 of PCV1 and PCV2, amino acid identity is that the intergenic diversity ratio of the ORF1 of 86%, two kind of genotype virus is less, has cross reaction between Rep albumen, and ORF2 genovariation is bigger, nucleic acid homology is 67%, and amino acid identity is 65% (Morozov et al., 1998), do not have cross reaction between Cap albumen, so the ORF2 gene can be used as the foundation of differential diagnosis.Discover that all include Poly (A) signal at the conservative section of PCV1 and PCV2, on normal chain, the Poly of PCV1 (A) lays respectively at: 314nt-319nt, 973nt-978nt; The Poly of PCV2 (A) lays respectively at: 327nt-332nt, 983nt-998nt; On minus strand, PCV1 has two Poly (A) signal to lay respectively at: 1015nt-1030nt, 1184nt-1189nt; And PCV2 has only a Poly (A) signal to be positioned at 1022nt-1027nt (Hamel et al., 1998).The ORF3 gene is the gene with critical function of recent studies in recent years, and it does not participate in the reproduction process of virus, and the Nonstructural Protein of coding virus can cause apoptosis, has vital role (Liu et al., 2005) in the PCV2 pathogenesis.

The Cap albumen of ORF2 genes encoding virus contains 234 amino acid, and the molecular weight size is 27.8kDa.Synthetic by genomic complementary strand.Cap albumen transcription initiation site is at 1238 Nucleotide, transcription product contains the non-coding leader sequence of being made up of 119 Nucleotide (1238nt-1120nt), the Cap promotor accurately is positioned 1353nt-1168nt Nucleotide zone, with the Rep gene overlapping (Mankertz et al., 1998) are arranged.Cap albumen can be expressed behind the PCV-II cells infected in 6-12 hour, was positioned at 12-24 hour (Meerts et al., 2005) in the nucleus.ORF2 gene variance ratio in PCV-II is bigger, and homology is about 70%.41 aminoacid sequence high conservatives of Cap albumen end, be rich in basic aminoacids (as arginine), the nuclear localization signal of coding virus, relevant with virus in endonuclear location, wherein 12-18 position and 34-41 amino acids residue to the location of ORF2 play crucial effects (Liu et al., 2001b).Mahe etc. (2000) find to co-exist in 5 antigen sites among the PCV, there is 1 to be arranged in ORF1 (185 aa-211 aa), all the other 4 all in ORF2, there are 2 to be the specific (B-121 of PCV2, B-133), have 1 all to react (B-146) with PCV1 and PCV2 antiserum(antisera), other has 1 not react with PCV1 and PCV2 antiserum(antisera) (B-152).Though the Cap albumen of PCV1 and PCV2 has the epitope of cross reaction to exist, no cross reaction exists between its ORF2, may be that the epitope that causes cross reaction in whole albumen is in the position that is difficult for being touched.Truong etc. (2001) have verified also that with immune dependent linearity B-cell epitope and ELISA method the 69-83 position of ORF2 and 117-131 (B-133 position) amino acids residue are the specific antigens determinants, wherein B-133 is one of SD significant antigenic determinant of PCV2, is used for the serodiagnosis of PCV2.

The virulence of ORF2 gene decision PCV2, the variation of ORF2 gene causes the difference of various places PCV2 strain isolated on virulence, and the ORF2 gene may have certain relation with the virus multiplication titre.Fenaux etc. (2003) utilize virus infection clones that the ORF2 of PCV1 and the ORF2 gene of PCV2 are exchanged, and find that the virulence of virus descends greatly.

Though the Cap albumen of PCV1 and PCV2 has the epitope that causes cross reaction to exist, and does not have cross reaction between the ORF2 of the two, so, all concentrate on ORF2 gene or the Cap albumen about the research of PCV2 diagnosis and recombinant vaccine.Mahe etc. (2000) propose to utilize PCV2-ORF2 polypeptide or intact proteins to set up the differential diagnosis method of difference PCV1 and PCV2 as diagnostic antigen.Blanchard etc. (2003) utilize the Cap albumen of PCV2 to set up as antigen and detect PCV2 specific antibody indirect ELISA, and comparing its accuracy rate can reach more than 90% with IFA, and has obtained good effect in clinical detection.Kamstrup etc. (2004) have studied the dna vaccination of ORF2 gene, and Blanchard etc. (2003b) have obtained the ideal immune effect with the Cap protein immunization pig of baculovirus expression.

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 at 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 pig circular ring 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 that obtains to express pig II type PCV-II major antigen site, and this reorganization bacterium contains pig II type PCV-II essential immunizing antigen Cap albumen.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 pig II type PCV-II bivalent genetic engineering vaccine; The 3rd purpose of the present invention is the application of above-mentioned reorganization bacterium in preparation Salmonella choleraesuls and pig II type PCV-II bivalent genetic engineering vaccine.

The present invention is achieved through the following technical solutions:

The applicant is by engineered method, obtain a kind of Salmonella choleraesuls (Salmonella choleraesuis) C501/pYA-Δ 41ORF2 of reorganization of the expression pig II type PCV-II 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 December 24th, 2009, and its deposit number is CCTCCNO:M209314.

The preparation method of a kind of recombinant salmonella choleraesuis C501/pYA-Δ 41ORF2, it comprises the following steps:

1) clone of PCV2 stoning signal for locating ORF2: with the recombinant plasmid pMD-PCV2 (Li Wenjie of agricultural microorganism National Key Laboratory of the Hua Zhong Agriculture University at applicant place preparation, what opening, Deng. the gene type assay [J] of Chinese some areas porcine circovirus 2 type. journal of animal science and veterinary medicine, 2009,40 (9): 1358-1362) be masterplate amplification Δ 41ORF2 fragment, clip size is 582bp, connect pMD-18T, transform DH5 α competent cell, the picking positive colony through PCR and enzyme cut identify show make up correct.More than 98%, there is not the base mispairing through the last sequence homology of issuing of check order Δ 41ORF2 sequence and GeneBank really;

2) structure of recombinant plasmid pYA-Δ 41ORF2: the PCR product that reclaims Δ 41ORF2 gene, and with restriction enzyme Saul I and Hind III double digestion, after purifying reclaims, connect and use the pYA3493 carrier of handling with quadrat method, Transformed E .coli χ 6097 competent cells obtain recombinant expression plasmid pYA-Δ 41ORF2.PCR and enzyme are cut the result and are shown that recombinant expression plasmid pYA-Δ 41ORF2 makes up.The sequencing result shows that sequence homology that the Δ 41ORF2 fragment inserted in the pYA-Δ 41ORF2 recombinant plasmid of structure and GeneBank go up issue more than 98%, any base mutation does not take place.

3) make up recombinant bacterial strain C501 (pYA-Δ 41ORF2): with the recombinant plasmid pYA-Δ 41ORF2 that builds, electricity is converted into Salmonella choleraesuls Δ asdC500 disappearance strain competent cell, and obtaining deposit number is the recombinant salmonella choleraesuis C501/pYA-Δ 41ORF2 of CCTCC NO:M209314.

The applicant obtains the gene fragment ORF2 (nucleotide sequence) in a kind of synthetic pig II type PCV-II essential immunizing antigen Cap albumen major antigen site, it is characterized in that its nucleotide sequence is (its preparation method is seen shown in the embodiment) shown in sequence table.

The applicant utilizes described recombinant salmonella choleraesuis C501/pYA-Δ 41ORF2 bacterium liquid to prepare successfully a kind of bivalent genetic engineering vaccine of preventing and treating Salmonella choleraesuls disease and pig circular ring virus 2 viral disease.

The recombinant salmonella choleraesuis strain in the above-mentioned expression pig II type PCV-II Cap albumen major antigen site that does not contain resistance marker lacked cell walls essential in the salmonella gene group 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-Δ 41ORF2 (containing the asd gene) simultaneously, has kept the immunological characteristic of parent strain C500 as the Salmonella choleraesuls attenuated vaccine strain.Plasmid pYA-Δ 41ORF2 can express asd gene and the proteic antigen site gene of pig II type PCV-II Cap in this bacterial strain.Wherein, the asd gene expression product provides salmonella strain essential cell walls forms relevant composition.The proteic antigen site gene expression product of Cap provides the good immunogenicity at pig II type PCV-II.

The recombinant salmonella choleraesuis strain C501/pYA-Δ 41ORF2 that does not contain the expression pig II type PCV-II major antigen site of resistance marker of the present invention, this engineering strain C501/pYA-Δ 41ORF2 is derived from China and has used the commercialization Salmonella choleraesuls attenuated vaccine strain C500 more than 50 years.Recombinant salmonella choleraesuis strain C501/pYA-Δ 41ORF2 of the present invention has lacked the asd gene in the C500 strain gene group, has added the plasmid pYA-Δ 41ORF2 that contains the proteic antigen site gene of pig II type PCV-II Cap simultaneously.Owing to need the recombinant bacterial strain that exists of pYA-Δ 41ORF2 to survive, therefore improved the stability of plasmid pYA-Δ 41ORF2 (Cap proteantigen locus gene just) in recombinant bacterial strain greatly.The stably express of Cap proteantigen locus gene in recombinant bacterial strain makes it to have at the good immune protective efficiency of pig II type PCV-II.

Basic construction method of the present invention is: the Salmonella choleraesuls attenuated vaccine strain C500 Δ asd Δ crp disappearance strain that utilizes the asd genetically deficient that the microorganism National Key Laboratory of the Ministry of Agriculture at the applicant place 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), reclaim the plasmid pYA-Δ 41ORF2 that contains pig II type PCV-II Cap proteantigen site simultaneously.41ORF2 changes in the C500asd gene-deleted strain with plasmid pYA-Δ, owing to growth need that plasmid pYA-Δ 41ORF2 and C500asd gene-deleted strain are complementary stablizes coexistence, form recombinant C 501 (the pYA-Δ 41ORF2) bacterial strain in the expression pig II type PCV-II Cap albumen major antigen site that does not contain resistance marker that we invent.The bivalent genetic engineering vaccine that can be used for preparing and pig circular ring virus 2 viral disease sick by the recombinant bacterial strain of a large amount of biological experiment digital proof the present invention preparations at Salmonella choleraesuls.

Major advantage of the present invention is:

1, the major antigen site is the main immunogenic gene fragment of pig II type PCV-II in the pig II type PCV-II Cap albumen of recombinant bacterial strain expression of the present invention, has good immune protection.Because the harm of pig II type PCV-II is serious day by day, and does not also have extensive stock pig II type PCV-II vaccine in the market.Therefore, the vaccine made from 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 at 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 at Salmonella choleraesuls and two kinds of cause of diseases of pig II type PCV-II 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 described.

Description of drawings

Sequence table SEQ ID NO:1 is the proteic nucleotide sequence of Cap that the present invention clones (insertion) pig II type PCV-II stoning signal for locating.

Fig. 1: the physical map that is the recombinant plasmid pYA-Δ 41ORF2 for preparing of the present invention.

Fig. 2: the PCR that is the recombinant plasmid pYA-Δ 41ORF2 for preparing of the present invention identifies electrophoretogram.

M:DNA Marker (DL2000) 1:ddH2O among the figure; 2:pYA-Δ 41ORF2; 3:pYA-Δ 41ORF2

Fig. 3: the enzyme that is the recombinant plasmid pYA-Δ 41ORF2 for preparing of the present invention is cut qualification result.

M1:DNA Marker among the figure (DL 2000, and DL 15,000) 1:pYA3493/Saul I+Hind III2And 3:pYA-Δ 41ORF2/Saul 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 pig II type PCV-II Cap albumen major antigen site that does not contain resistance marker among the present invention.M:DNA marker among the figure (DL 2,000); 1:ddH ₂The O negative control; 2: the recombinant plasmid positive control; 3-8:1-50 is for recombinant bacterial strain.

Fig. 5: the growth curve of recombinant bacterial strain C501 (pYA-Δ 41ORF2), empty plasmid control strain C500 (pYA3493) and parent strain C500.

Fig. 6: be the schema that the recombinant bacterial strain C501 (pYA-Δ 41ORF2) for preparing of the present invention makes up.

Fig. 7: the SDS-PAGE and the Western-blot analytical results that are a kind of recombinant salmonella choleraesuis living vaccine bacterial strain C501 (pYA-Δ 41ORF2) of the expression pig II type PCV-II Cap albumen major antigen site that does not contain resistance marker among the present invention.

Fig. 7 A detects fusion rotein Cpro-Δ 41ORF2 with the SDS-PAGE method, and 1 is C501 (pYA3493) among the figure; 2-7 is recombinant bacterial strain C501 (pYA-Δ 41ORF2); Arrow is depicted as amalgamation and expression PROTEIN C pro-Δ 41ORF2.

Fig. 7 B detects fusion rotein Cpro-Δ 41ORF2 with the Western-blot method, and 1-2 is C501 (pYA-Δ 41ORF2) among the figure; 3 is C501 (pYA3493) among the figure

Fig. 8: the collection of illustrative plates that is recombinant salmonella used carrier among the present invention.

Fig. 9: be Salmonellas serum antibody level.

Figure 10: be pig II type PCV-II Cap protein I gG antibody horizontal in the recombinant salmonella choleraesuis.

Embodiment

The present invention is further illustrated below in conjunction with Figure of description.

The segmental amplification of embodiment 1 pig II type PCV-II stoning signal for locating ORF2 protein gene

Design of primers (being used for gene clone and Molecular Detection)

Pig 2 type PCV-II sequence (accession number: AY035820) according to Gene Bank issue, design the primer (seeing Table 1) of 1 pair of stoning signal for locating ORF2 gene that can increase, pMD-PCV2 plasmid (the Li Wenjie that announces from this laboratory, what opening, Deng. the gene type assay [J] of Chinese some areas porcine circovirus 2 type. journal of animal science and veterinary medicine, 2009,40 (9): amplification stoning signal for locating ORF2 gene 1358-1362), clip size after the amplification is 582bp, and the primer upstream and downstream is introduced Saul I and Hind III restriction enzyme site respectively.This primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

The primer that table 1 the present invention is used

Structure and the evaluation of embodiment 2 recombinant plasmid pYA-Δ 41ORF2

Reclaim the PCR product of Δ 41ORF2 gene, and with restriction enzyme Saul I and Hind III double digestion, after purifying reclaims, connect the pYA3493 carrier of handling with restriction enzyme Saul I and Hind III double digestion (Dr.Roy Curtiss professor III of Washington, DC university is so kind as to give), Transformed E .coli χ 6097 competent cells obtain recombinant plasmid pYA-Δ 41ORF2.PCR and enzyme are cut the result and are shown that recombinant expression plasmid pYA-Δ 41ORF2 successfully constructs.The sequencing result shows that sequence homology that the Δ 41ORF2 fragment inserted in the pYA-Δ 41ORF2 recombinant plasmid of structure and GenBank go up issue all more than 98%, any base mutation does not take place.The physical map of recombinant expression plasmid pYA-Δ 41ORF2 and enzyme are cut qualification result (seeing Fig. 1, Fig. 3).

Embodiment 3 expresses structure and the evaluation of Cap protein gene segmental Salmonella choleraesuls recombinant bacterial strain C501 (pYA-Δ 41ORF2)

Press voltage 2.0Kv with BioRad GeneP μ lser II electroporation, time 4ms, the parameter of electric capacity 25F and pulse resistance 200 Ω transforms Δ asdC500 disappearance strain competent cell with recombinant plasmid pYA-Δ 41ORF2 electricity, screens positive recombinant on the negative flat board of DAP.Positive colony is because contain the ability that has expression DAP with the balance expression plasmid of asd gene complementation, so do not need to add DAP in the substratum again.Recombinant bacterial strain is carried out PCR identify, can amplify Δ 41ORF2 gene 581bp (Fig. 2), it is correct that the result shows that the reorganization bacterium makes up, and the applicant is with its called after C501 (pYA-Δ 41ORF2).

Embodiment 4 expresses the biological characteristics of Cap protein gene segmental Salmonella choleraesuls recombinant bacterial strain C501 (pYA-Δ 41ORF2)

1, the expression characterization analysis of C501 (pYA-Δ 41ORF2) recombinant bacterial strain

SDS-PAGE result shows, recombinant salmonella choleraesuis bacterial strain C501 of the present invention (pYA-Δ 41ORF2) has the obvious expression band at about 26kDa place, the Western-blot analysis revealed, the expressed albumen of recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2) can with pig 2 type PCV-II monoclonal antibody generation specific reactions, empty plasmid control strain C501 (pYA3493) then can not react (Fig. 7 B).

2, the phenotypic evaluation of recombinant bacterial strain C501 (pYA-Δ 41ORF2)

C500 is identical with parent plant, recombinant salmonella choleraesuis bacterial strain C501 of the present invention (pYA-Δ 41ORF2) can utilize maltose, the bacterium colony that takes on a red color on the maconkey agar flat board with maltose grows up to colourless, smooth, moistening oyster white bacterium colony on the LB flat board.Biochemical identification result shows, C501 (pYA3493) and C501 (pYA-Δ 41ORF2) and parent bacterium C500 all do not produce H2S, can not utilize lactose, sucrose, pectinose, melampyrum and urea, but can utilize maltose, glucose, rhamnosyl, seminose and wood sugar as sole carbon source.These results show that the biochemical characteristic of recombinant bacterial strain C501 of the present invention (pYA-Δ 41ORF2) is consistent with parent strain C500, meet the typical phenotypic characteristic of Salmonella choleraesuls.

3, the growth characteristics research of recombinant bacterial strain C501 (pYA-Δ 41ORF2)

From the growth curve (Fig. 5) of recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2), empty plasmid control strain C500 (pYA3493) and parent strain C500 as can be seen, the speed of growth is similar to parent strain C500 for recombinant bacterial strain C501 (pYA-Δ 41ORF2), and the speed of growth of empty plasmid control strain C500 (pYA3493) is more relatively slow than parent strain C500.

4, the genetic stability of recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2)

Recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2) was passed for 50 generations continuously in the LB liquid nutrient medium, getting the culture that contains identical CFU the 1st, 10,20,30,40 and 50 generations respectively is template, carries out PCR and detects the genetic stability of Δ 41ORF2 gene in C501.The result shows that the 1st, 10,20,30,40 and 50 generations all can amplify the band of 581bp, and its brightness does not have difference (Fig. 4).The culture that contains identical CFU of getting above generation respectively carries out Western-blot, all can get monoclonal antibody reactive with anti-PCV2-ORF2, and the result shows the secreting, expressing (Fig. 6 A) of reorganization bacterium C501 (pYA-Δ 41ORF2) stabilizability.The above results shows that recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2) can genetic stability.

Embodiment 5 recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2) detect at the intravital immune efficacy of mouse

1, the immune programme for children of mouse:

The BALB/c mouse of using 6-7 age in week is as the immune efficacy evaluation, be divided into 3 groups according to test requirements document, be respectively recombinant salmonella choleraesuis bacterial strain C501 (pYA-Δ 41ORF2) vaccine group, Salmonella choleraesuls 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 5.1 * 10 ⁹CFU viable bacteria amount) bacterium liquid or LB substratum, booster immunization is 1 time after 14 days.Exempt from every the mouse tail vein negative pressure blood sampling of 1,2,3,4 weeks of back respectively at preceding 0 day of immunity, head, collect serum, adopt indirect elisa method to detect the horizontal (see figure 9) (ELISA of Salmonellas serum antibody, working method is with reference to Xu Yindi. the structure of Salmonella choleraesuls C500 strain crp-, asd-disappearance strain balanced lethal system and application .[doctorate paper]. Wuhan: Hua Zhong Agriculture University Library, 2006.).The commodity ORF2 albumen bag that uses the production of preceding biological products limited liability company of Wuhan section simultaneously is by elisa plate (product batch number: 091005, date manufactured: 091015), (method is with reference to Liu Zhonghui etc. to press indirect ELISA method, immunology common experimental technology, Beijing: Science Press, 2002) detect recombinant C ap protein I gG antibody horizontal (see figure 10).

Embodiment 7 recombinant salmonella choleraesuis bacterium C501 (pYA-Δ 41ORF2) bivalent genetic engineering vaccine is at the intravital immune efficacy of pig

Since in July, 2009, successively at the recombinant salmonella choleraesuis bacteria vaccine of the present invention's preparation of four batches of A, B, C and D pig farm oral immunities.The recombinant salmonella choleraesuis bacteria vaccine (abbreviation bivalent genetic engineering vaccine) that A does not use on the pig farm the present invention to prepare is preceding, and the piglet case fatality rate is about 50%, and behind the oral bivalent genetic engineering vaccine of the present invention, survival rate is 90%.Behind other each oral bivalent genetic engineering vaccines of the present invention, also receive the effect similar to the A field.

Sequence table

＜110〉Hua Zhong Agriculture University

＜120〉recombinant salmonella choleraesuis and bivalent genetic engineering vaccine and preparation method

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<141>2009-12-20

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＜213〉pig circular ring virus (Porcine circovirus)

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Claims (4)

1. the pig II type PCV-II proteic recombinant salmonella choleraesuis of major antigen Cap (Salmonellacholeraesuis) C501 (pYA-Δ 41ORF2) is expressed in a strain, be deposited in Chinese typical culture collection center, its deposit number is: CCTCCNO:M209314.

2. the preparation method of a recombinant salmonella choleraesuis C501 (pYA-Δ 41ORF2), it comprises the following steps:

1) amplification pig II type PCV-II Cap albumen stoning signal for locating gene: with recombinant plasmid pMD-PCV2 is masterplate amplification Δ 41ORF2 fragment, connects pMD-18T, transforms DH5 α competent cell, and the picking positive colony carries out pcr amplification, obtains the PCR product;

2) construction recombination plasmid pYA-Δ 41ORF2: with the PCR product of the Δ 41ORF2 gene that reclaims with Sal I and Hind III double digestion, after purifying reclaims, connect pYA3493 carrier with Sal I and Hind III double digestion, Transformed E .coli χ 6097 competent cells obtain recombinant plasmid pYA-Δ 41ORF2;

3) make up recombinant bacterial strain C501 (pYA-Δ 41ORF2): with the recombinant plasmid pYA-Δ 41ORF2 that builds, electricity is converted into Salmonella choleraesuls Δ asd C500 disappearance strain competent cell, and obtaining deposit number is the recombinant salmonella choleraesuis C501/pYA-Δ 41ORF2 of CCTCC M209314.

3. a bivalent genetic engineering vaccine is characterized in that, is the bivalent genetic engineering vaccine that the recombinant salmonella choleraesuis C501/pYA-Δ 41ORF2 bacterium liquid of CCTCC NO:M209314 is prepared into Salmonella choleraesuls and pig II type PCV-II with deposit number.

4. the application of the described recombinant salmonella choleraesuis of claim 1 in preparation Salmonella choleraesuls and pig II type PCV-II bivalent genetic engineering vaccine.

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