Construction And Immunoprotection Evalution Of Orally Attenuated Recombinant Salmonella Gallinarum Vaccine Expressing SEF14 Fimbriae Of Salmonella Enteritidis

Salmonellosis is a common zoonotic disease caused by Salmonella.Except newborn animal,the poultry become asymptomatic carrier and long-term bacteriological positive after infection,resulting serious economic losses in poultry industry.Salmonella enteritidis(S.enteritidis)is one of the major causes of salmonellosis and food poisoning.The main method of preventing and controlling S.enteritidis in daily production is antibiotic treatment,but the problem of drug resistance and drug residues are suffered more and more attention due to the unreasonable use of antibiotics.Therefore,it is necessary for seeking for a safe and effective method to control S.enteritidis.Using vaccine combined with the rational use of antibiotics is the better effective way to prevent S.enteritidis infection.Therefore,it is significant to develop a broad spectrum of novel genetic engineering vaccine with strong protective ability.In this study,the attenuated Salmonella gallinarum(S.gallinarum)SG9R was used as the host strain and the asd gene was used as a marker of auxotrophic gene to construct attenuated S.gallinarum chromosome-plasmid lethal system,expressing SEF14 fimbriae antigens in S.enteritidis to investigate the immunoprotection effect of orally administered recombinant vaccine on the prevention of S.enteritidis.1.Cloning and expression of SEF14 fimbrial operon of Salmonella enteritidisExpression of SEF14 fimbriae is limited in S.enteritidis and other related D-group Salmonella serotypes.Our study is to clone and express gene cluster of SEF14 fimbriae in S.enteritidis in vitro,and study the bioactivity of the recombinant E.coli SE5000 expressing SEF14 fimbriae.SEF14 fimbrial operon gene clusters were amplified into two fragments by step-by-step PCR using the genomic DNA template of S.enteritidis wide type strain SD-2.The fimbrial operon gene cluster was amplified by inverse PCR and cloned into expression vector pBR322.The recombinant plasmid pBR322-sef containing the.sef gene clusters was constructed and screened correctly.The positive recombinant plasmid was transformed into non-fimbriae E.coli SE5000 strains to construct recombinant strain which was named as psef,pBR322 plasmid was transformed into E.coli SE5000 for negative control strain which was named p322.The recombination E.coli expressing SEF 14 fimbriae was tested by slide agglutination test.transmissible electromicroscope and Western blot.The results showed that the recombinant strain psef could undergo a significant agglutination reaction with the monoclonal antibody of SEF14,but not any visible aggulation reaction with p322.Expressed fimbriae on the surface of the recombinant bacteria psef was obversed through transmissible electromicroscope,Western blot analysis could obtain a protein band with a molecular weight of about 14.3kDa.These results indicated that SEF14 fimbriae was successfully expressed in SE5000.In this study,we established the basis for further study on the biology of Salmonella enteritidis SEF14 and the development of genetic engineering vector with SEF14 as an immunogen.2.Cloning and prokaryotic expression of the major subunit sefA of S.enteritidis SEF14 fimbrial operon and preparation of polyclonal antibodyAccording to the sequence of SEF14 fimbrial subunit sefA in S.enteritidis serotype in GenBank,specific primers were designed to amplify sefA genes by using S.enteritidis wild type strain SD-2 as the genomic DNA template.The PCR products with the NdeI and NotI restriction enzyme sites were inserted into the prokaryotic expression vector pET-28a(+)on the basis of the predetermined reading frame to construct the recombinant plasmid pET-28a(+)-sefA.Then the recombinant plasmid was transformed into E.coli BL21(DE3).SDS-PAGE demonstrated the recombinant expression vector could express the soluble protein with the size of about 14kDa.The recombinant protein had the highest expressional level when the recombinant strain was cultured at 0.2mM IPTG,25? for 5h.After purification by Ni-NTA affinity chromatography,the purified recombinant protein rSEFA was obtained to immunize the BALB/c mouse.The anti-rSEFA polyclonal antibody serum was raised and it could recognize S.enteritidis and the recombinant prokaryotic expression vector.In this study,we constructed and expressed high purified fusion recombinant protein rSEFA,and successful obtained the SEFA polyclonal antibody serum,laying a foundation for further study on the construction of recombinant genetically engineered vaccine which could express SEF14 fimbrial antigen.3.Construction and imunoprotection evalution of recombinant attenuated Salmonella Gallinarum expressing SEF14 fimbriae from S.enteritidisThe psef recombinant plasmid expressing SEF14 fimbriae was digested by SalI and NcoI to obtain the sef operon gene which was inserted into the expressing vector pYA3342 containing asd gene.The recombinant plasmid was transformed into SG9R?asd mutant to obtain recombinant attenuated S.gallinarum bacteria SG9R(pYA3342-sef).The subunit sefA gene of SEF 14 fimbriae was amplified by PCR with S.enteritidis standard strain SD-2 as the genomic DNA template,then we used the same method to construct the recombinant attenuated S.gallinarum bacteria SG9R(pYA3342-sefA).The expression of recombinant fimbriae was verified by slide agglutination test and Western blot.The phagocytosis test and survival test of Avain macrophage HD11 were carried out to study the function in the recombinant bacteria.At the same time,the biological characteristics such as growth characteristics,genetic stability and oral safety of recombinant bacteria were analyzed.All chickens could survive after orally inoculated with l×109cfu of two immunization groups when they were 35-day old,which confimed the safety of the recombinant vaccine.35-day old chickens were orally immuned with SG9R(pYA3342-sef),SG9R(pYA3342-sefA)and empty vector control group SG9R(pYA3342),while control group were injected with PBS.The expression of CD3 +,CD8 + T lymphocytes in peripheral blood was analyzed by flow cytometry.The levels of CD3 + and CD8 + T cells in SG9R(pYA3342-sef)group were significantly higher than those in other groups(P<0.05),and achieved the highest two weeks after second immunity.The serum levels of IgG in SG9R(pYA3342-sef)group were significantly higher than those in other groups range from a week after second immunity to two weeks after second immunity(P<0.05);The levels of specific sIgA were detected by indirect ELISA.The level of sIgA in the duodenum,jejunum,cecum and ileum of SG9R(pYA3342-sef)group was significantly higher than those in other groups two weeks after second immunity(P<0.05).Two weeks after second immunity,all chickens were challenged with S.enteritidis wide type strain SD-2.It was found that although the lethality of each group is 0,the SG9R(pYA3342-sef)group had a significant effect lower than other groups when 5 chicken of each group were dissected randomly.The results showed that the recombinant vaccine strain SG9R(pYA3342-sef)had a good immunoprotective effect on Salmonella enteritidis.