CN103013895B - Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application - Google Patents
Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application Download PDFInfo
- Publication number
- CN103013895B CN103013895B CN201110286965.8A CN201110286965A CN103013895B CN 103013895 B CN103013895 B CN 103013895B CN 201110286965 A CN201110286965 A CN 201110286965A CN 103013895 B CN103013895 B CN 103013895B
- Authority
- CN
- China
- Prior art keywords
- coe
- vaccine
- epidemic diarrhea
- porcine epidemic
- diarrhea virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention belongs to the technical field of animal bacterial genetic engineering, and concretely relates to a construction of recombinant Salmonella choleraesuis strains C501-Coe and C501-SD with no resistance marker and expressing main antigenic sites of porcine epidemic diarrhea virus, a preparation of a vaccine and an application. The invention obtains the recombinant Salmonella choleraesuis strains C501-Coe and C501-SD with no resistance marker and expressing the main antigenic sites of the porcine epidemic diarrhea virus, and the strains have the following accession number respectively: CCTCC NO: M2011296 and CCTCC NO: M2011297. The two recombinant strains are deleted with an asd gene necessary for growth of the S. choleraesuis, and contain plasmids which can express the asd gene, as well as a COE gene fragment and a SD gene fragment of the Porcine epidemic diarrhea virus in the strains. <{EN3}>The invention further discloses a method and an application by using the recombinant strain to prepare the vaccine of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. <{EN4}>The vaccine provided by the invention can stimulate swine to generate a protective immunization reaction for resisting the Salmonella choleraesuis and the Porcine epidemic diarrhea virus, and can effectively prevent infection of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. The invention further discloses a method and an application by using the recombinant strain to prepare the vaccine of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus. The vaccine provided by the invention can stimulate swine to generate a protective immunization reaction for resisting the Salmonella choleraesuis and the Porcine epidemic diarrhea virus, and can effectively prevent infection of the Salmonella choleraesuis and the Porcine epidemic diarrhea virus.
Description
Technical field
The invention belongs to animal bacteria gene engineering technology field, be specifically related to two kinds of construction and applications that do not contain the Salmonella choleraesuls living vaccine bacterial strain of the restructuring of expressing respectively Porcine epidemic diarrhea virus protective antigen gene-COE gene and SD gene of resistance marker.
Background technology
Salmonellas (Salmonellu) is the entozoic Gram-negative intestinal bacillis of a kind of born of the same parents, has aggressive, and modal salmonella has Salmonella typhimurium, Salmonella choleraesuls and Salmonella enteritidis etc.Salmonellas all has stronger virulence to humans and animals, can cause the gastrointestinal tract disease of human body and animal, and the viability of Salmonellas in external environment is stronger, utilize engineered method cause weak after, still there is stronger invasiveness.
The vaccine of initial prevention Salmonellas is full bacterium deactivation vaccine, but the humoral immunization that this vaccine can only excitating organism, and easily causes side effect, and in addition, also existing needs immunity repeatedly and without the shortcoming of cross protection, thereby can not widespread use.After this, researcher simulates the mode of Salmonellas enteron aisle natural infection, give the Salmonellas (as the oral weak malicious seedling of Salmonella enteritidis Ty21a) of the oral attenuation of animal, found that, can induce body to produce better humoral immunization and cellular immunization, therefore be subject to a lot of researchers' generally attention (Gernianier R et al., 1975).
Due to enteritis salmonella typhi Ty21a, to cause weak genetic background unintelligible, various countries researcher, attempt using engineered method, research genetic background is the weak virus gene deletion of vaccine strain of typhoid fever clearly, for oral immunity research, for example, aro gene-deleted strain (Dougan G et al., 1988), cya/crp gene-deleted strain (Alper M D et al., 1978), Dam and phoP/phoQ gene-deleted strain (Galan J E et al., 1989), asd gene-deleted strain (Xu Yindi etc., 2006) etc.
Weak malicious Salmonellas is except itself can be used as vaccine, also can be used as carrier and express exogenous antigen gene, in recent years, exogenous antigen taking weak malicious Salmonellas as vector expression tumour, virus, bacterium and parasite etc., development multivalent genetic engineered vaccine becomes the study hotspot in this field, thereby for development of new recombinant vaccine has been opened up new approach (Zhao Z et al., 2008).
In recent years, the recombinant vaccine that weak malicious Salmonellas is carrier becomes the focus of research, and its superiority is mainly manifested in (Xu draws younger brother's Ph D dissertation, 2006):
(1) validity of antigen presentation, when especially oral or collunarium is immune, effect better (Dietrich et al., 1998);
(2) can produce specific cytotoxic t lymphocytes and kill and wound reaction (CTL effect);
(3) can excite mucous membrane and systemic immunity reaction.After vaccine oral administration taking weak malicious Salmonellas as carrier or the mode immunity of collunarium, can directly exogenous antigen be delivered to the mucosa-associated lymphoid tissue (MALT) to body, thereby the mucous membrane of excitating organism and systemic immunity reaction (Spreng et al., 2006);
(4) there is immunoadjuvant function.The LPS of Salmonellas is a kind of inherent adjuvant, in addition, there are some researches show, there is a non-methylated CpG DNA sequence dna Salmonellas inside, there is stronger immunostimulatory activity, can promote that internal body Th type is main immunne response, thereby, be considered to have immunostimulant and the immunological adjuvant (Paglia et al., 1998) of application potential;
(5) immunity has persistence.The entrained plasmid of attenuation salmonella carrier is difficult for losing, and can be present in steadily in the long term in body Lymphoid tissue, thereby does not need the also immune response of excitating organism chronically of repeated multiple times ground booster immunization;
(6) combined immunization effect.The antigen that attenuation salmonella itself is entrained and the exogenous antigen of plasmid expression have played the effect of combined immunization, simultaneously, more than 2 or 2 exogenous antigen genes can also be inserted in expression plasmid, after immunity host, can produce corresponding immune response for different exogenous antigens, thereby reach the curative effect of primary immune response control various diseases;
(7) expressed antigen protein does not need to purify.Vaccine taking attenuation salmonella as carrier, only needs incubated overnight to get final product direct immunization, thereby has saved the step of inductor induction and purifying protein, and preparation process is simple, save time, and is easy to store transport, therefore, can significantly reduce vaccine production cost;
(8) vaccination ways is easy, and easy handling is suitable for herd immunity, and security is good.
Salmonella choleraesuls C500 low virulent strain is containing in the substratum of thaliium acetate, continuous passage causes weak bacterial strain for 500 times, it has good immunogenicity, practice has also proved that C500 strain has good immunogenicity, and can activate whole body, part and mucomembranous immune system, have the advantages that side effect is low, security is good.Wherein, that the most frequently used is Salmonellas Asd-balanced lethal system (Galan J E, et al.1990).State Key Laboratory of Agricultural Microbiology Xu Yindi doctor has built the asd gene-deleted strain Δ asd C500 of C500 strain, confirm the immunogenicity of Δ asd C500 gene-deleted strain through experiment, compared with C500 bacterial strain, virulence is lower, better (the Xu Yindi etc. of security, 2006), and be applied to the preparation of live recombined vaccines, as applicant place agricultural microorganism National Key Laboratory has built the recombinant salmonella living vaccine of transmissible gastroenteritis of swine, through clinical verification, this vaccine has good immunoprophylaxis effect, and apply for Chinese invention patent (number of patent application is 2009100639279).
Porcine epizootic diarrhea (PED) is caused by Porcine epidemic diarrhea virus (PEDV), a kind of acute infectious disease of height contact, after pig infects PEDV, often cause the acute enteritis of piglet and mortality watery diarrhea until dehydration is dead, its Clinical symptoms is mainly manifested in piglet vomiting, severe diarrhea and dehydration; PEDV infected pigs, cuts open atrophy and come off (Pensaert and Yeo, 2006) of the intestinal villus of jejunum that inspection pathological change main manifestations is pig and ileal segment.PED is very large to the harm of pig, and the pig at various ages all can fall ill, but the most serious to the harm of sucking piglets, and 20 ages in days reach more than 95% (Timoney J F et al., 1988) with interior piglet mortality ratio, bring heavy economic losses to pig industry.
At present, PED is worldwide widely current.At European Countries, PED popularity degree than before obviously reduces, but the infection rate of PEDV is very high in pig farm, Asia, especially in Korea S, Japan and Chinese etc., from pathogenesis and clinical symptom, PEDV is very similar to TGEV, and because its infection rate is apparently higher than TGEV and porcine rotavirus (PoRV), cause huge loss (Cavanagh D et al., 1997) to pig industry.
PEDV belongs to coronaviridae, coronavirus genus.PED finds and reports to be the Britain (Oldham, 1972) in 1971 first.Henceforth, this disease was reported (Puranaveja et al., 2009) on a large scale in Europe and Asia, from 1976, China has reported the generation of PED in succession, and PED has become one of important pig virus diarrhoea disease popular in world wide now.1978, cause pathogenic agent-PEDV that this is sick, obtain first qualification (Chasey et al. in Britain and Belgium, 1978), and be listed in possible the member of coronavirus genus in (ICTV) the 5th time report of ICTV in 1991, until the full member who is just listed in coronavirus genus is reported in the 6th time of nineteen ninety-five.
PEDV particle shape feature and other member's of coronaviridae very similar (Chasey et al., 1978), Spike Glycoprotein (S albumen), membrane glycoprotein (M albumen) and envelope glycoprotein (E albumen) are distributed in the surface of virus particle; Nucleocapsid protein (N albumen) is positioned at virus particle inside, the nucleocapsid structure of PEDV is interacted and is formed by N albumen and geneome RNA, this makes PEDV and Transmissible gastroenteritis virus on morphology, be difficult to difference (Pensaert et al., 1986).So far, research finds that PEDV only exists a serotype (Witte et al., 1981).
PEDV S albumen plays a significant role in mediated infection host produces the process of neutralizing antibody, in recent years, and the research that researchist has successively carried out PEDV S gene both at home and abroad.Chang in 2002 etc. are according to the neutralizing epitope sequence of TGEV S gene, infer an epitope district (499～638aa) that PEDV S gene, and called after COE, confirm through test, this gene has good immunogenicity, and also as target gene, be applied to (Nguyen et al., 2009 among the preparation of Transgenic Plant Vaccines and recombinant vaccine; Ge Junwei etc., 2010).PEDV is taking humoral immunization as main virus, thereby, B cell antigen epi-position has great importance in anti-PEDV infects, the people such as Sun (2008) identify the S1D5 (744-759aa) and the S1D6 (756-771aa) that in S1D (636-789aa) district of learning S1 subunit, comprise by experiment, may be two B cell epitopes (Sun D B et al. of PEDV, 2008), S1D district has also comprised the good high-affinity epitope sequences of an immunogenicity S1P3 (697-742aa) in addition.
The method that conventional prevention PED occurs is both at home and abroad that by the intestinal contents of the ight soil of PEDV infected pigs or morbidity piglet, artificial challenge pregnant sow, stimulates and produce maternal antibody, after piglet is sucked the breast, obtains protection, reduces mortality ratio and shortens the popular time of PED.
Identical with most of virus diseases, to the prevention and control of PED, be mainly to put prevention first with vaccine immunity.At present, the business-like vaccine of PED is mainly two kinds of deactivation vaccine and weak malicious seedlings, and two kinds of vaccines are after use, although can prevent to a certain extent the generation of PED, effect is undesirable.Infer that its former because PEDV is mainly through intestinal tract infections pig, in the immunologic process that local Mucosal Immunity system infects at anti-PEDV, bring into play important effect (Pospischil A et al., 2002), thereby stimulate the specificity sIgA of intestinal mucosa generation, become the problem of anti-this disease of system generation and development PED vaccine key.But existing commercialization deactivation vaccine and weak malicious seedling, after immunity, can only be induced humoral immunoresponse(HI), can not activate mucosa-immune system, even if there is higher serum antibody titer, still can not stop in cause of disease per mucous membrane invasion body, thereby immune effect is not good.At present, though the correlative study of existing PED Transgenic Plant Vaccines and genetically engineered living vaccine, just in the experimental study stage, also unrealized commercialization, therefore, develop safer, efficient, cheap new generation vaccine and be world's pig industry in the urgent need to.
In view of attenuation salmonella, to have as vaccine carrier the efficiency of transporting high, and immunization method is simple, and immune effect is better, can excite the advantage such as general immunity and local mucosal immunity simultaneously, has good application prospect.There are a lot of defects in the conventional construction method of recombinant salmonella vaccine, carries the expression plasmid of resistant gene as previously used, because of its existing Biosafety problem, and do not accepted by people.Salmonella choleraesuls C500asd plasmid-carrier balanced lethal system that this institute is used has advantages of non-resistant mark, in addition, also have that immunogenicity is good, expression amount is high, exogenous gene expression is stable and do not need the advantages such as purifying, thereby the live recombined vaccines that is developed as expression Porcine epidemic diarrhea virus major antigen gene has broad application prospects.
Summary of the invention
The object of the invention is to overcome the defect that prior art exists, its first object is to obtain the recombinant salmonella choleraesuis strain of expressing Porcine epidemic diarrhea virus protective antigen protein gene-COE gene and SD protein gene.
Second object of the present invention is to utilize above-mentioned recombinant bacterium to obtain a kind of Salmonella choleraesuls and Porcine epidemic diarrhea virus bivalent genetic engineering vaccine.
The 3rd object of the present invention is the application of above-mentioned recombinant bacterium in preparation Salmonella choleraesuls and Porcine epidemic diarrhea virus bivalent genetic engineering vaccine.
The present invention is achieved through the following technical solutions:
Applicant is by engineered method, the Salmonella choleraesuls (Salmonella choleraesuis)-C501-COE and the C501-SD that express the restructuring of Porcine epidemic diarrhea virus protective antigen gene-COE gene and SD gene are obtained, this two bacterial strain delivers on August 22nd, 2011 Chinese Typical Representative microbial preservation center (CCTCC) preservation that is positioned at Wuhan City, Hubei Province Wuhan University, and its deposit number is respectively CCTCCNO:M2011296; CCTCC NO:M2011297.
The Salmonella choleraesuls C501-COE of two restructuring and the preparation method of C501-SD, mainly comprise the following steps:
1) structure of prokaryotic expression plasmid pET-32a-COE and pET-32a-SD: from suffering from Porcine Epidemic Diarrhea (Porcine epidemic diarrhea, PED) in sick swine excrement pathological material of disease, amplification Porcine epidemic diarrhea virus COE gene and SD gene, and respectively by its subclone to prokaryotic expression carrier pET-32a, build prokaryotic expression plasmid pET-32a-COE and pET-32a-SD;
2) structure of recombinant plasmid pYA-COE and pYA-SD: carry out double digestion by building correct prokaryotic expression plasmid, and connect with the shuttle plasmid pYA3493 through identical double digestion, transform the intestinal bacteria x6097 of Asd-, screening positive clone;
3) the Salmonella choleraesuls C501-COE of restructuring and the structure of C501-SD: recombinant plasmid pYA-COE and pYA-SD electricity are converted in Salmonella choleraesuls C500 Δ crp Δ asd gene-deleted strain, obtain deposit number and be respectively CCTCC NO:M2011296; Salmonella choleraesuls C501-COE and the C501-SD of the restructuring of CCTCC NO:M2011297.
Applicant obtains gene fragment COE gene, the SD gene in two kinds of Porcine epidemic diarrhea virus major antigen sites, and their nucleotide sequence is as shown in sequence table SEQ ID NO:1 and SEQ ID NO:3 (its preparation method is shown in shown in embodiment).
Applicant utilizes the Salmonella choleraesuls C501-COE bacterium liquid of described restructuring, Salmonella choleraesuls C501-SD bacterium liquid and the gelatin protective material of restructuring successfully to prepare a kind of bivalent genetic engineering vaccine of preventing and treating Salmonella choleraesuls and porcine epizootic diarrhea according to proper volume than (seeing embodiment).
The above-mentioned recombinant salmonella choleraesuis strain that does not contain the expression Porcine epidemic diarrhea virus major antigen site of resistance marker has lacked cell walls formation genes involved asd gene essential in salmonella strain genome and (need to be present in the inner or outer source of thalline containing the plasmid of asd gene and add ability normal growth on the substratum of DAP, breeding), this two strains recombinant bacterial strain contains respectively exogenous plasmid pYA-COE and pYA-SD (all containing asd gene) simultaneously, this two strains recombinant bacterial strain has all retained the immunological characteristic of parent strain C500 as Salmonella choleraesuls attenuated vaccine strain.Plasmid pYA-COE and pYA-SD all can express asd gene in this bacterial strain, and both can express respectively COE antigen site and SD antigen site gene in Porcine epidemic diarrhea virus S albumen.Wherein, asd gene expression product provides salmonella strain essential cell walls forms relevant composition.COE antigen site and SD antigen site gene expression product provide the good immunogenicity for Porcine epidemic diarrhea virus.
Recombinant salmonella choleraesuis strain C501-COE and the C501-SD that does not contain the expression Porcine epidemic diarrhea virus major antigen site of resistance marker of the present invention, this two strain gene engineerings bacterial strain is all derived from China and has used more than 50 years commercialization Salmonella choleraesuls attenuated vaccine strain C500.Described recombinant salmonella choleraesuis strain C501-COE of the present invention and C501-SD, lack the asd gene in C500 strain gene group, added the COE antigen site that contains in Porcine epidemic diarrhea virus S albumen and plasmid pYA-COE and the pYA-SD of SD antigen site gene simultaneously.Because the recombinant bacterial strain that exists that needs pYA-COE and pYA-SD could be survived, therefore greatly improve plasmid pYA-COE and pYA-SD (namely COE antigen site and the SD antigen site gene) stability in recombinant bacterial strain.COE antigen site and the stably express of SD antigen site gene in recombinant bacterial strain, make it to have for the good immune protective efficiency of transmissible gastro-enteritis virus.
Basic construction method of the present invention is: utilizing the Salmonella choleraesuls attenuated vaccine strain C500 Δ crp Δ asd gene-deleted strain (Xu Yindi etc., 2006) of the asd genetically deficient that the agriculture microorganism National Key Laboratory at the applicant place builds to build to contain can prokaryotic expression Porcine epidemic diarrhea virus COE and plasmid pYA-COE and the pYA-SD of SD gene.By recombinant plasmid respectively electricity proceed in C500asd gene-deleted strain; recombinant plasmid and C500asd gene-deleted strain form complementation like this; and meet growth needs; and then stable coexisting, form recombinant bacterial strain C501-COE and the C501-SD that can express Porcine epidemic diarrhea virus protective antigen that do not contain resistance marker that we invent.The recombinant bacterial strain of preparing by a large amount of biological experiment digital proof the present invention can be used for the bivalent genetic engineering vaccine of preparation for Salmonella choleraesuls and porcine epizootic diarrhea.
Major advantage of the present invention is:
1, the N in A, D antigen site and N albumen in the pig infectious gastroenteritis virus S protein that recombinant bacterial strain of the present invention is expressed
321antigen site is the important immunogenic gene fragment of transmissible gastro-enteritis virus; Porcine epidemic diarrhea virus protective antigen gene-COE gene and SD gene that recombinant bacterial strain of the present invention is expressed are the important immunogenic gene fragments of Porcine epidemic diarrhea virus, all have good immune protective.Because transmissible gastro-enteritis virus and pig, epidemic diarrhea virus harm are day by day serious, and in the market extensive stock transmissible gastroenteritis of swine and porcine epizootic diarrhea vaccine (being mainly adjuvant inactivated vaccine and weak malicious seedling) immune effect generally poor.Therefore, there is wide market application foreground with the vaccine that this engineering strain is made.
2, recombinant bacterial strain of the present invention is derived from China and has used more than 50 years commercialization Salmonella choleraesuls attenuated vaccine strain C500, has retained the immune efficacy of C500 for Salmonella choleraesuls completely.And, this recombinant bacterial strain virulence than C500 slightly a little less than, there is better biological safety.
3, recombinant bacterial strain of the present invention can provide the protection for Salmonella choleraesuls, Transmissible gastroenteritis virus and three kinds of cause of diseases of porcine epizootic diarrhea simultaneously.
4, the vaccine that the present invention develops, compares with weak malicious seedling with existing deactivation vaccine, have preparation process simple, save time, and be easy to store the feature of transport, significantly reduced the cost of manufacture of vaccine.
5, recombinant bacterial strain of the present invention, not containing resistance marker, meets the requirement of vaccine biological safety completely.
More detailed technical scheme is shown in described in embodiment.
Brief description of the drawings
Sequence table SEQ ID NO:1 is the nucleotide fragments of the Porcine epidemic diarrhea virus protective antigen gene COE that clones of the present invention.
Sequence table SEQ ID NO:3 is the nucleotide fragments of the Porcine epidemic diarrhea virus protective antigen gene SD that clones of the present invention.
Fig. 1: prepared by the present invention can prokaryotic expression porcine epizootic diarrhea protective antigen gene fragment build schema.
Fig. 2: prepared by the present invention can the COE gene of prokaryotic expression and the sequence alignment figure of SD gene.Wherein, Fig. 2 A: can the COE gene of prokaryotic expression and comparing of other strain corresponding sequence; Fig. 2 B: can the SD gene of prokaryotic expression and comparing of other strain corresponding sequence.
Fig. 3: the present invention prepares the collection of illustrative plates of recombinant salmonella shuttle plasmid pYA3493 used.
Fig. 4: the present invention prepares the PCR qualification picture of recombinant plasmid pYA-COE and pYA-SD.Wherein:
Fig. 4 A:pYA-COE PCR identifies picture.Wherein, M:DNA Marker (DL2000); 1-2:pYA-COE; 3: positive control; 4: negative control;
Fig. 4 B:pYA-SD PCR identifies picture.Wherein, M:DNA Marker (DL2000); 1-2:pYA-SD; 3: positive control; 4: negative control.
Fig. 5: the present invention prepares the enzyme of recombinant plasmid pYA-COE and pYA-SD and cuts qualification result electrophorogram.Wherein, M1:DNA Marker (DL2000); M2:DNA marker (DL15000); 1:pYA3493/EcoR I; 2:pYA-COE/EcoRI+HindIII; 3:pYA-SD/EcoRI+Sal I.
Fig. 6: recombinant bacterium C501-COE prepared by the present invention and the structure schema of C501-SD.
Fig. 7: the PCR qualification figure of recombinant bacterium C501-COE prepared by the present invention and C501-SD.Wherein,
Fig. 7 A: recombinant bacterium C501-COE PCR identifies picture.Wherein, M:DNA Marker (DL2000); 1-2:C501-COE; 3: positive control;
4: negative control;
Fig. 7 B: recombinant bacterium C501-SD PCR identifies picture.Wherein, M:DNA Marker (DL2000); 1-2:C501-SD; 3: positive control;
4: negative control.
Fig. 8: the amplification picture of Salmonellas invA gene in recombinant bacterial strain C501-COE prepared by the present invention and C501-SD.Wherein,
M:DNA Marker (DL2000); 1: positive control; InvA gene amplification in 2:C501-COE; InvA gene amplification in 3:501-SD.
Fig. 9: utilize Western blotting method to detect the secreting, expressing of albumen in the recombinant bacterium C501-COE for preparing of the present invention and C501-SD.Wherein:
Fig. 9 A: contain the secreting, expressing of COE albumen in recombinant bacterium C501-COE.Wherein, M:Prestained Protein Ladder;
Fig. 9 B: contain the secreting, expressing of SD albumen in recombinant bacterium C501-SD.Wherein, M:Prestained Protein Ladder.
Figure 10: utilize indirect immunofluorescence experiment to detect the location of expressing protein in bacterium in the recombinant bacterium C501-COE for preparing of the present invention and C501-SD.Wherein, Figure 10 A:C501-COE; Figure 10 B:C501-SD; Figure 10 C:C501-pYA.
Figure 11: recombinant bacterium C501-COE prepared by the present invention and the growth curve chart of C501-SD.
Figure 12: recombinant bacterium C501-COE prepared by the present invention and the genetic stability experimental result of C501-SD.Wherein:
Figure 12 A: the genetic stability qualification result of recombinant bacterium C501-COE.Wherein, M:DNA Marker (DL2000); 1-6: be respectively that recombinant bacterium C501-COE goes down to posterity 50 times, 40 times, 30 times, 20 times, 10 times and the PCR qualification result of 1st generation;
Figure 12 B: the genetic stability qualification result of recombinant bacterium C501-SD.Wherein, M:DNA Marker (DL2000); 1-6: the PCR qualification result that is respectively recombinant bacterium C501-SD 1st generation and goes down to posterity after 20 times, 30 times, 40 times and 50 times.
Figure 13: recombinant bacterium C501-COE prepared by the present invention and C501-SD after immune BalB/C mouse for exogenous antigen mucous membrane sIgA antibody horizontal.Wherein:
Figure 13 A: the mucous membrane sIgA antibody horizontal of the anti-COE albumen after recombinant bacterium C501-COE immune mouse;
Figure 13 B: the mucous membrane sIgA antibody horizontal of the anti-SD albumen after recombinant bacterium C501-SD immune mouse.
Figure 14: recombinant bacterium C501-COE prepared by the present invention and C501-SD after immune BalB/C mouse for the serum IgG antibody level of exogenous antigen.Wherein:
Figure 14 A: the serum IgG antibody level of the anti-COE albumen after recombinant bacterium C501-COE immune mouse;
Figure 14 B: the serum IgG antibody level of the anti-SD albumen after recombinant bacterium C501-SD immune mouse.
Figure 15: the antibody horizontal of recombinant bacterium C501-COE prepared by the present invention and C501-SD pin Salmonellas after immune BalB/C mouse.Wherein:
Figure 15 A: the serum IgG antibody level of the anti-salmonella after recombinant bacterium C501-COE and C501-SD immune mouse;
Figure 15 B: the mucous membrane sIgA antibody horizontal of the anti-salmonella after recombinant bacterium C501-COE and C501-SD immune mouse.
Figure 16: the level of difference comparison on the IFN-γ mRNA that recombinant bacterium C501-COE prepared by the present invention and C501-SD induce in Mice Body.Wherein,
Figure 16 A: the IFN-γ mRNA level of recombinant bacterium C501-COE induction contrasts the results of comparison of bacterium with empty plasmid;
Figure 16 B: the IFN-γ mRNA level of recombinant bacterium C501-SD induction contrasts the results of comparison of bacterium with empty plasmid.
Figure 17: the diversity ratio of the IFN-γ protein expression level that recombinant bacterium C501-COE prepared by the present invention and C501-SD induce in Mice Body.Wherein:
Figure 17 A: the IFN-γ protein expression level of recombinant bacterium C501-COE induction contrasts the results of comparison of bacterium with empty plasmid;
Figure 17 B: the IFN-γ protein expression level of recombinant bacterium C501-SD induction contrasts the results of comparison of bacterium with empty plasmid.
Embodiment
Below in conjunction with Figure of description, the present invention is further illustrated.
Embodiment 1: preparation can the porcine epizootic diarrhea protective antigen COE of prokaryotic expression and the gene fragment of SD albumen
(1) prepare the required design of primers of prokaryotic expression plasmid pET-32a-COE and pET-32a-SD, its sequence is as described in Table 1.
Table 1 is prepared prokaryotic expression plasmid pET-32a-COE and the required primer sequence of pET-32a-SD
In table 1, the underscore part of primer is restriction enzyme site.
(2) preparation can prokaryotic expression COE and SD fragment
Utilize Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and the pig A rotavirus multiple RT-PCR detection method (Zhang Kun, 2010) of a female report, detect the clinical pathological material of disease of censorship.The positive ight soil pathological material of disease infecting from Porcine epidemic diarrhea virus, extract geneome RNA (adopting the total RNA extraction reagent box of BioFlux company, the test kit specification sheets providing according to the said firm operation), then carry out reverse transcription.Taking cDNA that reverse transcription was obtained as template, respectively taking the EP1/HP2 shown in table 1 and SDE1/SDS2 as primer, pcr amplification is with the PEDV COE gene of EcoR I, HindIII restriction enzyme site with the SD gene fragment of ECOR I, Sal I restriction enzyme site, and amplification system is as described below:
The pcr amplification condition of COE gene segment and SD gene fragment: after 95 DEG C of denaturation 4min, circulate as follows: 94 DEG C of sex change 40s, 58.1 DEG C of annealing 45s, 72 DEG C are extended 50s, 30 circulations, last 72 DEG C of 10min.
After having increased, get PCR product 8 μ L, add 1 μ L10 × Loading Buffer, carry out electrophoresis with 0.8% sepharose, EB dyeing, observes electrophoresis result.After electrophoresis, reclaim with BioFlux company glue the purifying (according to the specification sheets operation of this test kit) that test kit carries out goal gene.COE gene fragment after purifying and prokaryotic expression carrier pET-32a are carried out respectively running glue recovery after EcoR I and HindIII double digestion, utilize T4DNA ligase enzyme to connect the rear escherichia coli expression Host Strains BL21 of conversion (DE3); SD gene fragment after purifying and prokaryotic expression carrier pET-32a are carried out respectively running glue recovery after EcoR I and Sal I double digestion, utilize T4DNA ligase enzyme to connect the rear escherichia coli expression Host Strains BL21 of conversion (DE3).Through PCR, double digestion and order-checking qualification, finally obtain can prokaryotic expression porcine epizootic diarrhea COE and SD gene fragment.The preparation flow of gene fragment that can prokaryotic expression as shown in Figure 1.The comparison result of sequencing result and other strain sequence as shown in Figure 2.
Embodiment 2: the Construction and identification of recombinant plasmid pYA-COE and pYA-SD
Will be with EcoR I, the recombinant plasmid pET-32a-COE of HindIII restriction enzyme site and with ECOR I, the pET-32a-SD of Sal I restriction enzyme site carries out double digestion recovery, then connect with the same shuttle plasmid pYA3493 (Fig. 3 is shown in by plasmid construction collection of illustrative plates) reclaiming through double digestion, connect the intestinal bacteria x6097 (source: Dr.Roy Curtiss professor III of Washington, DC university is so kind as to give) that product transforms asd genetically deficient, screening positive clone, extract plasmid, through PCR and double digestion qualification, obtain building correct recombinant plasmid pYA-COE and pYA-SD (as shown in Figure 4 and Figure 5).Wherein, pcr amplification system and amplification condition are as embodiment 1, described in (2).
Embodiment 3: express Porcine epidemic diarrhea virus COE and the recombinant salmonella bacterial strain C501-COE of SD fusion rotein and the Construction and identification of C501-SD
(1) design of the required primer of the Salmonellas C501-COE of qualification restructuring and C501-SD, its DNA sequence dna is as shown in table 2:
The DNA sequence dna of the required primer of the Salmonellas C501-COE of table 2 qualification restructuring and C501-SD
(2) the Salmonellas C501-COE of restructuring and the Construction and identification method of C501-SD
By the correct recombinant plasmid pYA-COE of qualification and pYA-SD (embodiment 2 is seen in source) respectively electricity be converted in the C500-competent cell of asd disappearance, electroporation (Bio-Rad GenePulserII) parameter is set to: voltage 2.2Kv, electric capacity 25 μ F, pulse resistance 200 Ω and time 4ms, build schema and see Fig. 6.On the negative flat board of DAP, picking list bacterium colony is cultivated 8-10h in TSB liquid nutrient medium, draws 1mL bacterium solution preparation template, taking the EP1/HP2 shown in table 1 and SDE1/SDS2 as primer, carries out the amplification (as shown in Figure 7) of object fragment respectively; The Auele Specific Primer pi1/pi2 (in table 2) that re-uses Salmonellas specific gene invA gene (GenBank:M90846.1) carries out Salmonellas specificity identification (as shown in Figure 8) to recombinant bacterium; The last qualification of checking order taking the primer pY1 shown in table 2 as upstream sequencing primer.It is correct that result shows that the Salmonella choleraesuls of the restructuring that contains pYA-COE and pYA-SD plasmid build, applicant is respectively by its called after Salmonella choleraesuls bacterial strain (Salmonella choleraesuis) C501-COE and C501-SD, and delivers the preservation of Chinese Typical Representative culture collection center (CCTCC) for patented procedure.
Embodiment 4: express Porcine epidemic diarrhea virus COE and the recombinant salmonella bacterial strain C501-COE of SD fusion rotein and the biological characteristics of C501-SD
(1) the expression characterization analysis of recombinant bacterium C501-COE and C501-SD
Single bacterium colony of picking recombinant bacterium C501-COE and C501-SD is in TSB liquid nutrient medium respectively, 37 DEG C, 200r/min cultivates 12h, places 12h for 4 DEG C, is inoculated in TSB liquid nutrient medium (formula: 3%TSB powder (30g) is dissolved in ddH by the volume ratio of 1: 100
2in O, after 15 pounds of autoclaving 15min in room temperature preservation.) in, 37 DEG C, 200r/min, cultivate 6-8h, the centrifugal 10min of 8000rpm, collects respectively thalline and supernatant, supernatant liquor is through 0.22 μ m membrane filtration, get 900 μ l supernatants, and add 100% trichoroacetic acid(TCA) 100 μ l, make the final concentration of above-mentioned recombinant bacterium all reach 10%, after ice bath 30min, 12000r/min, centrifugal 30min, abandons supernatant; Add the absolute ethanol washing precipitation of 1mL precooling, 12000r/min, centrifugal 10min, so repeated washing 2 times.Obtained albumen (is so kind as to give by Harbin Veterinary Medicine Inst., China Academy of Agriculture with pig source porcine epidemic diarrhea resisting virus-positive serum, see described in genetic resources table) make primary antibodie, carry out Western blotting qualification (as shown in Figure 9), result shows that recombinant bacterium of the present invention can secreting, expressing fusion rotein, and fusion rotein can with the Porcine epidemic diarrhea virus positive serum antibody generation specific reaction of pig.
In addition, through indirect immunofluorescence experiment, fusion rotein is identified the location in bacterium, authentication method is with reference to Master's thesis (the Wang Miao .2006 of Wang Miao.), experimental result demonstration, the expressed fusion rotein of recombinant bacterium is positioned the surface (as shown in figure 10) of bacterium.
(2) the growth characteristics analysis of recombinant bacterium C501-COE and C501-SD
By recombinant bacterium C501-COE, C501-SD in TSB after 37 DEG C of overnight incubation, carry out continuous 10 times of dilutions, choose 3 suitable dilution gradients, each extent of dilution is got 100 μ L and is uniformly coated on TSA flat board, and each gradient is respectively done 3 repetitions, 37 DEG C, overnight incubation, counting, averages, and calculates the CFU (colony-forming unit) of stoste.It is 10 that switching makes its final concentration
6cFU/mL, 37 DEG C, 200r/min shaking culture, at interval of 1h sampling, surveys OD600 value, draws growth curve.Identical method is drawn the growth curve of parent plant pig Salmonellas C500 and empty carrier plasmid control strain C501-pYA, then compares the difference of its growth characteristics.Can find out from the growth curve (as shown in figure 11) of recombinant bacterium, recombinant bacterium C501-COE, recombinant bacterium C501-SD, empty carrier plasmid control strain C501-pYA are in culturing process, growth conditions is substantially similar to parent strain pig Salmonellas C500, and just the speed of growth is slightly slower than parent pig Salmonellas.
(3) phenotypic evaluation of recombinant bacterium C501-COE and C501-SD
Respectively by recombinant bacterium C501-COE, C501-SD, empty plasmid control strain C501-pYA and parent strain C500 streak inoculation in TSA flat board, then picking list bacterium colony, the carbon source biochemical identification pipes such as pectinose, seminose, hydrogen sulfide, lactose, wood sugar, urea, glucose, rhamnosyl, melampyrin, sucrose of transferring respectively, 37 DEG C, incubation 24h, carries out the phenotype of recombinant bacterium and judges.Biochemical identification result shows, recombinant bacterium C501-COE, C501-SD are identical with parent bacterium C500 biochemical characteristic with C501-pYA, all can not utilize sucrose, pectinose, urea, lactose, melampyrin and sucrose, but can utilize seminose, wood sugar, glucose and rhamnosyl as sole carbon source.
(4) genetic stability of recombinant bacterium C501-COE and C501-SD
By recombinant bacterium C501-COE and C501-SD with TSA solid medium (formula: 4%TSA powder (40g) is dissolved in ddH2O, after 15 pounds of autoclaving 15min in room temperature preservation.) streak culture on the flat board prepared, picking list bacterium colony is to liquid nutrient medium, 37 DEG C, 200r/min, shaking culture 12h, then be transferred in fresh TSB liquid nutrient medium by the volume ratio of 1: 100, cultivate 12h, repeating so continuously switching cultivates 50 times, with the 1st, 10, 20, 30, the culture in 40 and 50 generations is template, carry out pcr amplification with the primer EP1/HP2 described in table 1 and SDE1/SDS2 respectively, pcr amplification system and amplification condition are as embodiment 1, (2) described in, detect recombinant plasmid pYA-COE and the genetic stability of pYA-SD in Δ asd C500.Qualification result proves, all can from each generation recombinant bacterium culture, amplify the specific DNA band (as shown in figure 12) of 480bp and 462bp.
Embodiment 5: the immunogenicity of analyzing recombinant bacterium taking Balb/C mouse as animal model
1. mouse immune experimental design
70 BALB/c mouse are (purchased from Disease Prevention Control Center, Hubei Prov, see described in genetic resources table) be divided at random 7 groups, every group 10, be respectively oral recombinant bacterium C501-COE, intramuscular injection recombinant bacterium C501-COE, oral recombinant bacterium C501-SD, intramuscular injection recombinant bacterium C501-SD, oral recombinant bacterium C501-pYA, intramuscular injection recombinant bacterium C501-pYA and TSB control group, after within two weeks and two, exempting from after head exempts from, (head exempts from latter 4 weeks) docking in two weeks is taken a blood sample and collects intestinal contents, indirect ELISA method detects antibody horizontal (IgA and IgG antibody), concrete operation step is as described below:
1.1 immune serum IgG antibody horizontal ELISA detect:
According to document " detailed annotation of vaccine gordian technique " the original work second edition (A. Robinson etc., 2006), prokaryotic expression protein (COE albumen and SD albumen) is pressed to the concentration coated elisa plate in 0.5 μ g/ hole, 100 μ l/ holes, 4 DEG C, coated spending the night, washings washing 3 times, 3min/ time;
(2) add confining liquid to seal, 150 μ l/ holes, 37 DEG C, sealing 2h, takes out washing 3 times, 3min/ time;
(3) serum to be checked was carried out to doubling dilution since 1: 2 or 1: 10, add enzyme plate, 100 μ l/ holes, 37 DEG C, reaction 1h, takes out washing 3 times, 3min/ time;
(4) add the sheep anti mouse HRP-IgG of 1: 6000 times of dilution, 100 μ l/ holes, 37 DEG C, reaction 1h, washs 3min/ time 5-6 time;
(5) add tetramethyl benzidine substrate (A+B) 100 μ l, room temperature lucifuge reaction 10-15min, then add 50 μ l 0.25%HF termination reactions, measures OD630 by microplate reader
nm;
(6) select OD630
nm>=0.2 reacting hole, as the positive, gets the inverse of the highly diluted multiple of serum to be checked, is converted into the titre of antibody.
1.2 immune mouse intestinal secretion IgA antibody horizontals detect:
Respectively at 21d after immunity, 28d gets 2-3 mouse at random, and cervical vertebra dislocation is put to death, and scraping enteron aisle surface mucus is in 1.5ml centrifuge tube, and with 10 times of dilutions of pH 7.4PBS do, after mixing ,-20 DEG C save backup.
Enteron aisle sIgA antibody ELISA detection method is with described in embodiment 5,1.1.Wherein, composition and the preparation process of indirect ELISA detection method core reagent used are as follows:
The carbonate buffer solution (25mmol/L) of coating buffer: pH9.6; Na
2cO
31.59g, NaHCO
32.93g, ddH
2o is settled to 1L;
PBS damping fluid: 140mmol NaCl (8.18g), 2.7mmol KCl (0.20g), 10mmol Na
2hPO
4(3.58g), 1.8mmol KH
2pO
4(0.245g), be dissolved in 800mL ddH
2in O, be adjusted to after pH7.4 ddH with the NaOH of 5mol/L
2o is settled to 1L;
Washings: add 0.05% Tween-20 in PBS damping fluid;
Confining liquid: add 0.50g bovine serum albumin (BSA) in 1L washings;
The phosphate-citrate salts damping fluid 25.7mL of substrate buffer solution: pH5.0, the citric acid solution 24.3mL of 0.1mol/L, the Na2HPO4 25.7mL of 0.2mol/L, ddH2O 50mL;
TMB mother liquor: 0.2g TMB dry powder is dissolved in the dehydrated alcohol of 100mL;
Substrate solution (TMB): by TMB mother liquor with substrate buffer solution in after mixing in the ratio of 1: 20, in every milliliter of substrate solution, add 30% H2O2 0.2 μ L;
Stop buffer: 0.25%HF.
Before immunity, need carry out live bacterial count to recombinant bacterium.4h before oral group of immunity, the BALB/c mouse material of need cutting off the water, the oral 50 μ l 10%NaHCO of 20min before immune
3in and hydrochloric acid in gastric juice, then use BALB/c mouse gavage pin oral immunity 200 μ l No. 12, make every BALB/c mouse immunizing dose be about 1.5 × 10
8individual viable bacteria amount (dosage reference: People's Republic of China's veterinary biologics quality standard, 2001 editions); Every BALB/c mouse immunizing dose of intramuscular injection group is identical with under oral immunity group; TSB blank group immunization 200 μ l.
Latter 2 weeks of immunity, chooses at random 5 for every group and carries out booster immunization.Respectively within 2 weeks, carrying out mouse tail vein negative pressure hemostix behind 2 weeks, booster immunization before first immunisation, after first immunisation, collect serum, by 5 BALB/c mouse serum balanced mix, adopt indirect elisa method to detect each group of average antibody level.
2. the detection of the porcine epidemic diarrhea resisting of immune BALB/c mouse virus protective antigen protein antibodies level
The BALB/c mouse serum of gather respectively before immunity, head exempting from latter 2 weeks after exempting from for 2 weeks and two utilizes indirect ELISA method to detect serum IgG antibody; Respectively at 21d after immunity, 28d gets 2-3 BALB/c mouse at random, and cervical vertebra dislocation is put to death, and scraping enteron aisle surface mucus, in 1.5ml centrifuge tube, with pH 7.4PBS dilution, utilizes indirect ELISA method to detect mucous membrane sIgA antibody.According to document " detailed annotation of vaccine gordian technique " the original work second edition (A. Robinson etc., 2006), prokaryotic expression protein (COE albumen and SD albumen) is pressed to the concentration coated elisa plate in 0.5 μ g/ hole, serum to be checked or intestinal mucosa content were carried out to doubling dilution since 1: 2 or 1: 10, join respectively in respective reaction hole and detect.
Detected result is as follows: 1. sIgA production, recombinant bacterium C501-COE and C501-SD oral immunity group sIgA antibody titers are respectively 1: 1280 and 1: 2048, and the sIgA antibody titers of intramuscular injection group is respectively 1: 640 and 1: 1024 (as shown in figure 13); 2. serum IgG antibody production, IgG antibody titers in recombinant bacterium C501-COE and C501-SD intramuscular injection group is respectively 1: 4096 and 1: 2560, and the IgG antibody titers that oral immunity group produces is respectively 1: 2048 and 1: 1280 (as shown in figure 14)
(3) the horizontal ELISA of immune BALB/c mouse antibodies toward salmonella detects
The preparation of Salmonellas ELISA antigen: the single bacterium colony of picking Salmonellas is in 5mL TSB liquid nutrient medium, 37 DEG C, activation is spent the night, be to be forwarded to 100mL TSB liquid nutrient medium at 1: 100 by volume ratio, 37 DEG C, 200r/min, cultivate 8h, 8000r/min, centrifugal 10min, collect thalline, with the PBS washing of pH 7.4 2 times, then, with the PBS of pH 7.4 taking volume ratio as 1: 10 times concentrated resuspended, (UP 200S processor for ultrasonic wave-German Dr.Hielscher company manufactures to limpid in ultrasonication on ice, power: 200W, amplitude: 60%, operating frequency: 24KHz, the ultrasonic disruption time: 30s/ time, interval time: 1min/ time), 12000r/min, centrifugal 20min, abandon precipitation.With spectrophotometric determination supernatant protein concentration, packing 500 μ l/ pipes, save backup in-20 DEG C.According to document " detailed annotation of vaccine gordian technique " the original work second edition (A. Robinson etc., 2006), by the Salmonellas albumen of preparation, by the coated elisa plate of the concentration in 0.5 μ g/ hole, indirect ELISA method detects the Specific IgA antibody level in IgG antibody and the intestinal mucosa in immune BALB/c mouse serum.
Detected result demonstration, recombinant bacterial strain C501-COE and C501-SD immune group BALB/c mouse are all induced the Salmonellas serum IgG antibody and the enteron aisle sIgA antibody (as shown in figure 15) that have produced with empty carrier control strain C501-pYA immune group BALB/c mouse similar level
(4) cellular immunization detects
Concrete grammar is pressed document (Xiao Shaobo, 2004) and is carried out:
Prepare mouse spleen lymphocyte, and to adjust cell concn be 2 × 10
6/ mL, joins in 24 porocyte culture plates, and 1mL/ hole, makes stimulated in vitro with the target protein of purifying former, and to make final concentration of protein be 10 μ g/mL.24 porocyte culture plates are put in to 37 DEG C, 5%CO
2in cell culture incubator, cultivate, after 20h, respectively select a cell hole, extract cell total rna, measure the difference in IFN-γ mrna expression level by the quantitative method of relative fluorescence.Remaining cell hole, after 72h, collecting cell supernatant, utilizes two sandwich ELISA methods (to adopt the R & D mouse IFN-γ of company detection kit to detect, concrete operation step is with reference to the specification sheets of this test kit), detect the difference on IFN-γ protein expression level.
Utilize the method for SYBR Green Real-time PCR, detect the mRNA of IFN-γ in BALB/c mouse immune spleen cell with respect to the expression amount of the mRNA of β-actin, and carry out relative quantitative assay, result shows, in recombinant bacterium C501-COE and C501-SD immune group BALB/c mouse splenocyte, the relative expression quantity of IFN-γ mRNA is higher than empty carrier plasmid C501-pYA immune group, and the relative expression quantity of the IFN-γ mRNA of intramuscular injection group will be higher than oral immunity group (as shown in figure 16).
Adopt two sandwich ELISA methods (to adopt the R & D mouse IFN-γ of company detection kit to detect, concrete operation step is with reference to the specification sheets of this test kit) detect the IFN-γ level of each immune group splenocyte secretion, result shows, in recombinant bacterium C501-COE of the present invention and C501-SD immune group BALB/c mouse splenocyte, IFN-γ protein expression level is apparently higher than higher than empty carrier plasmid C501-pYA immune group, and the horizontal outline of recombinant bacterium intramuscular injection group IFN-gamma antibodies is higher than oral immunity group.(as shown in figure 17)
In addition, the IFN-γ that Salmonellas empty plasmid control group C501-pYA induces be mRNA level or on protein level higher than TSB control group all, this may can to serve as adjuvant relevant with Salmonellas itself, thereby can stimulate body to produce more intense cell immune response.
Embodiment 6: utilize Salmonellas C501-COE and the C501-SD of restructuring to prepare bivalent genetic engineering vaccine
By streak culture on TSA solid medium to recombinant bacterium C501-COE and C501-SD, picking list bacterium colony is cultivated in TSB liquid nutrient medium, is cultured to viable bacteria concentration and reaches 2.5 × 10
10cFU/mL.By bacterium liquid low-speed centrifugal collection bacterium, the bacterium liquid in Salmonellas C501-COE and C501-SD: gelatin protective material (volume: volume) is that the ratio of 2: 1 adds gelatin protective material (protective material compound method: every 100mL ddH
2in O, add sucrose 40g, gelatin 8g; After fully dissolving in 121 DEG C of autoclaving 30min, save backup), in sterilizing freeze-drying bottle, press the packing of 2.0mL/ bottle, put freeze-drying in-50 DEG C of freeze driers, freeze-drying 36-40h rear pressing cover, dissolves and carries out live bacterial count (CFU) with physiological saline or PBS (pH7.4), and guaranteeing there is no living contaminants, be placed in-20 DEG C and save backup, as the vaccine strains of development recombiant vaccine.
Embodiment 7: the preliminary clinical protection effect observation of recombiant vaccine bacterial strain
(1) safety testing of recombiant vaccine bacterial strain to newborn piglet
Randomly draw the healthy 1 age in days piglets of 2 nests (kind is landrace) from a large scale of pig farm field, with 3 × 10
9the immunizing dose of CFU, freeze-drying attenuation salmonella vaccine prepared by oral immunity the present invention, tracing observation 7 days, finds that immunity front and back piglet body temperature is unchanged, and piglet can normally suck breast milk, illustrate that thus freeze-drying attenuation salmonella vaccine prepared by the present invention is safe.
(2) the immunoprotection test of the freeze-drying attenuation salmonella vaccine that prepared by the present invention to piglet
The large-scale pig farm of selecting two PEDV to infect, with 3 × 10
9the immunizing dose of CFU, freeze-drying attenuation salmonella vaccine prepared by oral immunity the present invention, tracing observation 1 week, records piglet (kind is landrace) sickness rate and surviving rate after the immunity of each pig farm, evaluates immune effect (in table 3 and table 4).
The immune effect of Porcine epidemic diarrhea virus recombinant salmonella choleraesuis genetically engineered living vaccine prepared by the A application the present invention of table 3 pig farm
The immune effect of Porcine epidemic diarrhea virus recombinant salmonella choleraesuis genetically engineered living vaccine prepared by the B application the present invention of table 4 pig farm
From the clinical trial statistic data of table 3 and table 4, not only security is better for freeze-drying attenuation salmonella vaccine prepared by the present invention, and has good preventive effect.After newborn piglet immunity recombiant vaccine, A field diarrhea rate drops to 18%～30%, B field mortality ratio by 100% and drops to 8%～20% by 80%, and result has shown that this recombiant vaccine has good immunoprophylaxis effect.
Reference
1.A. Robinson etc. the detailed annotation of vaccine gordian technique. the original work second edition. (Che Yanchun etc. translate for Li Qihan, Liu Longding). Beijing: Chemical Industry Press, 2006,12-14.
2. Ge Jun is big, Wang Miao, Li Yijing etc. express the immunne response [J] of the recombinant lactobacillus casei immune mouse induction of Porcine epidemic diarrhea virus neutralizing antigenic site. and Chinese veterinary science, 2010,40 (07): 708-712.
3. Wang Miao. express in Porcine epidemic diarrhea virus and the master of the structure Northeast Agricultural University Diplomarbeit .[master thesis of antigenic region Recombinant Lactococcus lactis system]. Wuhan: Library of Hua Zhong Agriculture University, 2006.
4. Xu draws younger brother, Guo Aizhen, Liu Weihong etc. structure and the qualification [J] of Salmonella choleraesuis C500 strain Δ crp Δ asd gene-deleted strain balanced lethal carrier system. and biotechnology journal, 2006,22 (3): 366-372.
5. Xu draws younger brother. Salmonella choleraesuis C500 strain crp
-, asd
-the construction and application .[doctorate paper of gene-deleted strain balanced lethal system]. Wuhan: Library of Hua Zhong Agriculture University, 2006.
6. the few ripple of Xiao. and Pseudorabies virus gC gene " the immune-enhancing effect research .[doctorate paper of Suicidal DNA Vaccine and VP22 protein transduction]. Wuhan: Library of Hua Zhong Agriculture University, 2004.
7. female, what opening. foundation and the clinical application [J] of Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus and pig A rotavirus multiple RT-PCR detection method. journal of animal science and veterinary medicine, 2010,41 (8): 1001-1005.
8.Alper?M?D，Ames?B?N.Transport?of?antibiotics?and?metabolite?analogs?by?systems?under?cyclic?AMP?control：positive?selection?of?Salmonella?typhimurium?cya?and?crp?mutants[J].J?Bacteriol，1978，133(1)：149-157.
9.Cavanagh?D，Brian?D?A，Briton?P，et?al.Nidovirales：a?new?order?comprising?Coronaviridae?and?Arteriviridae[J].Arch?Virol，1997，142(3)：629-633.
10.Chasey?D，Cartwright?S?F.Virus-like?particles?associated?with?porcine?epidemic?diarrhea.Res?Vet?Sci，1978，25(2)：255-256.
11.Chen?H.，Schifferli?D.M，et?al.Enhanced?immune?responses?to?vira?lepitopes?by?combining?macrophage-inducible?expression?with?multimeric?display?on?a?Salmonella?vector.Vaccine，2001，19(20-22)：3009-3018.
12.Dietrich?G，Bubert?A，Gentschev?I，et?al.Delivery?of?antigen-encoding?plasmid?DNA?into?the?cytosol?of?macrophages?by?attenuated?suicide?Listeria?monocytogenes[J].Nat?Biotechnol，1998，16(2)：181-185.
13.Dougan?G，Chatfield?S，Pickard?D，Bester?J，O’Callaghan?D?and?Maskell?D.Construction?and?characterization?of?vaccine?strains?of?Salmonella?harboring?mutations?in?two?different?aro?genes[J].J?Infect?Dis，1988，158(6)：1329-1335.
14.Galan?J?E?and?Curtiss?III?R.Virulence?and?vaccine?potential?of?phoP?mutants?of?Salmonella?typhimurium[J].Microb?Pathogen，1989，6：433-443.
15.Galan?J?E，Nakayama?K，Curtiss?R?D.Cloning?and?characterization?of?the?asd?gene?of?Salmonella?typhimurium：use?in?stable?maintenance?of?recombinant?plasmids?in?Salmonella?vaccine?strains[J].Gene，1990，94：29-35.
16.Gernianier?R，Furer?E.Isolation?and?characterization?of?GalE?mutant?Ty?21a?of?Salmonellu?typhi：a?candidate?strain?fora?live，oral?typhoid?vaccine[J].J?Infect?Dis，1975，131：553-558.
17.Nguyen?X?H，Young?S?K，Sang?C?J，et?al.Production?of?a?Heat-labile?Enterotoxin?B?Subunit-porcine?Epidemic?Diarrhea?Virus-neutralizing?Epitope?Fusion?Protein?in?Transgenic?Lettuce(Lactuca?sativa)[J].BIOTECHNOLO?BIOPROC?E，2009，14：731-737.
18.Paglia?P，Medina?E，Arioli?I，et?al.Gene?transfer?in?dendritic?cells，induced?by?oral?DNA?vaccination?with?Salmonella?typhimurium，results?in?protective?immunity?against?a?murine?fibrosarcoma[J].Blood，1998，92(9)：3172-3176.
19.Pensaert?M?B.Porcine?Epidemic?Diarrhea.DiseasesofSwine[J].Iowa?State?University?Press，1986，402-406.
20.Pensaert?M?B，Yeo?S?G.Porcine?epidemic?diarrhea.In：Straw?B?E，Zim-merman?J?J，D’Allaire?S，Taylor?D?J.(eds.)，Diseases?of?Swine.Wiley-Blackwell，Ames，2006，9：367-372.
21.Pospischil?A，Stuedli?A，Kiupel?M，et?al.Update?on?porcine?epidemic?diarrhea[J].J?Swine?Health?Prod，2002，10(2)：81-85.
22.Spreng?S，Dietrich?G，Weidinger?G.Rational?design?of?Salmonella-based?vaccination?strategies[J].Methods，2006，38：133-143.
23.Sun?D?B，Feng?L，Shi?H?Y，Chen?J?F，et?al.
cation?of?two?novel?B?cell?epitopes?on?porcine?epidemic?diarrhea?virus?spike?protein[J].Veterinary?Microbiology，2008，131：73-81.
24.Timoney?J?F，GillesPie?J?H，Seott?F?W，et?al.Microbiology?and?Infectious?Diease?of?Domestie?Animals.Ithaca：Comstock?Publishing?Associates，1988，897-898.
25.Zhao?Z，Xue?Y，Wu?B，et?al.Subcutaneous?vaccination?with?attenuated?Salmonella?enterica?serovar?Choleraesuis?C500?expressing?recombinant?filamentous?hemagglutinin?and?pertactin?antigens?protects?mice?against?fatal?infections?with?both?S.enterica?serovar?Choleraesuis?and?Bordetella?bronchiseptica[J].Infect?Immun，2008，76(5)：2157-2163
Claims (2)
1. Salmonella choleraesuls and porcine epizootic diarrhea bivalent genetic engineering vaccine, it is characterized in that, Salmonella choleraesuls (Salmonella choleraesuis) the C501-COE vaccine strain that this vaccine contains restructuring, it is deposited in Chinese Typical Representative culture collection center, preserving number is CCTCC NO:M2011296, this vaccine strain contains expresses Porcine epidemic diarrhea virus protective antigen gene COE, the sequence of its protein is as shown in sequence table SEQ ID NO:2, meter by volume, the bacterium liquid of described Salmonella choleraesuls C501-COE and the protectant ratio of gelatin are 2:1.
2. preserving number is Salmonella choleraesuls (Salmonella choleraesuis) C501-COE of the CCTCC NO:M2011296 application in preparation Salmonella choleraesuls and porcine epizootic diarrhea bivalent genetic engineering vaccine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110286965.8A CN103013895B (en) | 2011-09-23 | 2011-09-23 | Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110286965.8A CN103013895B (en) | 2011-09-23 | 2011-09-23 | Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103013895A CN103013895A (en) | 2013-04-03 |
| CN103013895B true CN103013895B (en) | 2014-07-16 |
Family
ID=47963012
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110286965.8A Active CN103013895B (en) | 2011-09-23 | 2011-09-23 | Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103013895B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103387998B (en) * | 2013-07-01 | 2015-05-20 | 北京市农林科学院 | Method for cultivating transgene corn that can express COE protein stably and special gene thereof |
| CN104667296A (en) * | 2015-02-04 | 2015-06-03 | 四川农业大学 | Vaccine for preventing porcine epizootic diarrhea |
| CN104673807A (en) * | 2015-02-04 | 2015-06-03 | 四川农业大学 | Recombinant salmonella containing porcine epidemic diarrhea virus S499-789 gene |
| CN104630240A (en) * | 2015-02-04 | 2015-05-20 | 四川农业大学 | Recombinant Salmonella comprising porcine epidemic diarrhea virus S499-650 gene |
| CN105002197A (en) * | 2015-07-06 | 2015-10-28 | 河南科技大学 | Nonresistant bifunctional DNA vaccine vector, and construction method and application thereof |
| CN105154377B (en) * | 2015-07-17 | 2020-02-11 | 河南科技大学 | Recombinant salmonella pullorum, preparation method and application |
| CN110093357B (en) * | 2019-04-17 | 2023-03-28 | 仲恺农业工程学院 | Multi-epitope antigen of porcine epidemic diarrhea virus, encoding gene, preparation method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030086062A (en) * | 2002-05-03 | 2003-11-07 | 장용석 | Transformants expressing epitope protein of porcine epidemic diarrhea virus and edible vaccine composition containing the same |
| CN101157907A (en) * | 2007-09-27 | 2008-04-09 | 华中农业大学 | Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof |
| CN101880647A (en) * | 2009-09-11 | 2010-11-10 | 华中农业大学 | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application |
| KR20110000854A (en) * | 2009-06-29 | 2011-01-06 | (주)애드바이오텍 | Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis |
| CN101979501A (en) * | 2010-09-17 | 2011-02-23 | 华中农业大学 | Recombinant Salmonella choleraesuis for expressing surface antigen gene sao of streptococcus suis type 2, vaccine and application |
-
2011
- 2011-09-23 CN CN201110286965.8A patent/CN103013895B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030086062A (en) * | 2002-05-03 | 2003-11-07 | 장용석 | Transformants expressing epitope protein of porcine epidemic diarrhea virus and edible vaccine composition containing the same |
| CN101157907A (en) * | 2007-09-27 | 2008-04-09 | 华中农业大学 | Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof |
| KR20110000854A (en) * | 2009-06-29 | 2011-01-06 | (주)애드바이오텍 | Composition of food additives of piglets containing igy from egg yolk for preventing of porcine epidemic diarrhea or transmissible gastroenteritis |
| CN101880647A (en) * | 2009-09-11 | 2010-11-10 | 华中农业大学 | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application |
| CN101979501A (en) * | 2010-09-17 | 2011-02-23 | 华中农业大学 | Recombinant Salmonella choleraesuis for expressing surface antigen gene sao of streptococcus suis type 2, vaccine and application |
Non-Patent Citations (6)
| Title |
|---|
| 满晓营等.表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性.《微生物学报》.2009,(第04期), |
| 猪霍乱沙门氏菌ΔasdC500株的生物学特性及作为活疫苗表达载体的应用;赵战勤等;《生物工程学报》;20090125(第01期);29-36 * |
| 表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性;满晓营等;《微生物学报》;20090404(第04期);518-523 * |
| 表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性;马丰英等;《微生物学报》;20110904(第09期);1270-1277 * |
| 赵战勤等.猪霍乱沙门氏菌ΔasdC500株的生物学特性及作为活疫苗表达载体的应用.《生物工程学报》.2009,(第01期), |
| 马丰英等.表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性.《微生物学报》.2011,(第09期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103013895A (en) | 2013-04-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103013895B (en) | Genetic engineering live vaccine of recombinant Salmonella choleraesuis and Porcine epidemic diarrhea virus, preparation and application | |
| LeCureux et al. | Lactobacillus mucosal vaccine vectors: immune responses against bacterial and viral antigens | |
| Mohamadzadeh et al. | Targeting mucosal dendritic cells with microbial antigens from probiotic lactic acid bacteria | |
| Tao et al. | A concise review of vaccines against Mycoplasma hyopneumoniae | |
| Huang et al. | Construction and immunogenicity analysis of Lactobacillus plantarum expressing a porcine epidemic diarrhea virus S gene fused to a DC-targeting peptide | |
| CN102281895A (en) | A vaccine for porcine post-weaning diarrhea caused by enterotoxigenic escherichia coli | |
| CN101880647B (en) | Recombinant salmonella choleraesuis, bivalent genetic engineering vaccine and application | |
| CN101157907B (en) | Recombinant salmonella choleraesuis strain for expression of pig origin bordetella bronchisepatica fhaB and prn gene segment, bacterin and uses thereof | |
| CN100537749C (en) | Express the reorganization bacterium of enterotoxin colibacillus adhesin gene and the application in Yolk antibody feed thereof | |
| CN102274496B (en) | O/Asia I type foot and mouth disease virus bivalent genetic engineering polypeptide vaccine, its preparation method and its purpose | |
| Cai et al. | Oral immunization with surface immunogenic protein from Streptococcus agalactiae expressed in Lactococcus lactis induces protective immune responses of tilapia (Oreochromis niloticus) | |
| CN102732473B (en) | The recombinant salmonella choleraesuis of expression mycoplasma hyopneumoniae p46 albumen and preparation method and application | |
| US9358283B2 (en) | Diatom-based vaccines | |
| CN105154377B (en) | Recombinant salmonella pullorum, preparation method and application | |
| CN105754905A (en) | Pseudomonas aeruginosa of minks and application of pseudomonas aeruginosa | |
| CN102741414B (en) | By recombination yeast oral/method of mucosal vaccination vaccine | |
| CN103421729B (en) | Gene recombined swine cholera salmonella choleraesuis vaccine for blue-ear disease and application thereof | |
| CN101962625A (en) | Salmonella choleraesuis gene deletion mutant without resistant marker and vaccine thereof | |
| CN103421732B (en) | Express the Salmonella choleraesuls attenuated vaccine of haemophilus parasuis surface antigen | |
| CN102732442A (en) | Salmonella choleraesuis double-gene-deletion strain free of resistance marker and application thereof | |
| CN103421733B (en) | A kind of haemophilus parasuis-Salmonella choleraesuls bigeminy gene engineering vaccine | |
| CN102380095A (en) | FMD trivalence polypeptide vaccine and preparation method and application thereof | |
| CN101838627A (en) | Recombination salmonella choleraesuis and bivalent genetic engineering vaccine and preparation method | |
| Jiao et al. | Immunization effect of recombinant Lactobacillus casei displaying Aeromonas veronii Aha1 with an LTB adjuvant in carp | |
| CN101037664A (en) | Bacterin for pleuropneumonia actinobacillus serotype 1 double-gene deletion mutant without resistance marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 430070 1 Lion Rock street, Hongshan District, Wuhan, Hubei Co-patentee after: WUHAN KEQIAN BIOLOGICAL CO., LTD. Patentee after: Huazhong Agricultural University Address before: 430070 1 Lion Rock street, Hongshan District, Wuhan, Hubei Co-patentee before: Wuhan Keqian Animal Biological Products Co., Ltd. Patentee before: Huazhong Agricultural University |
|
| CP01 | Change in the name or title of a patent holder |