Study Of Monovalent Candidate Vaccine Strains Against Streptococcus Suis Type Ⅱ With Its Virulence Mrp Gene

Streptococcus suis H is one of the most pathogenic and danger of streptococcus suis typese. It could lead to a series of inflammation which lead to death. Genetic engineering vaccine is most safe, low cost and effective method, easy to promote on a large scale in means of many epidemic preventions to streptococcus suis2. Muramidase released protein (MRP) is one of the first confirmed SS2's pathogenic factors. MRP has the most obvious immunogenicity. This reserch use this important virulence factor as immune original. And this reserch also study on enterotoxigenicEscherichia coli heatliable toxin gene (elt). At the same time a plasmid-chromosome balanced-lethal system was used to conduct a SS2's MRP candidate recombinant vaccine strains without antibiotics resistance genes. This experiment is divided into three parts.Firstly using broth medium in anaerobic conditions cultivture SS2sichuan separation strain. Isolate the muramidase released protein gene of Streptococcus suis serotypes2by PCR with the Streptococcus suis serotypes2strain from Sichuan in China, Ligate it to the pMD18-T vector. Kpn I digest recombinant vector to make sure the insert direction is correct. After choose the right insert plasmid using Pvu I digest it and Recover the5889bp fragment. At the same time, amplify asd gene from plasmid pZLZl. The insert it to recombinant vector and the antibiotic resistance gene ampr of the vector was destroyed by insertion of as a gene. Then the recombinant vector was successively transformed into E.coli X6097, Salmonella X3O37(asd+) and Salmonella X4072(asd). A plasmid-chromosome balanced-lethal system was conducted. The SDS-PAGE results indicated MRP was expressed by Salmonella X4072.The second part is cloning the complete elt gene from plasmid pMM085by designing specific primers with new restriction sites. Then double digest it and pGEX6-P-1expression vector. Let1231bp and4771bp fragments in one by a ligase. The PCR detection indicated that restructuring plasmid was successfully built.The last part is construct a recombinant expression plasmid ofmrp and eltB gene. Get the mrp gene from SS2train adding two restriction site MlsI and Sall. Product was be directly amplified to T vector, name this restructuring plasmid pTM. Then double digest pTM and pGEX6-P-1expression vector by MlsI and SalI.Recovery1231bp and4771bp fragments and link them, name the new restructuring plasmid as pZMO1. At the same time, clone the complete eltB gene from plasmid pMM085by designing specific primers with new restriction sitesMluI and ApaI. Recovey and let the407bp and8286bp fragments in one by a ligase, name the new restructuring plasmid as pZM02. The result of PCR mrp gene and eltB gene from pZM02indicated that recombinant plasmid was successfully built.This research lays the foundation for further study.