Cloning,Expression And Preliminary Immunological Features Of The Myophilin From Trichinella Spiralis Muscle Larva

Trichinosis is a foodborne zoonotic disease caused by trichinella spiralisthat can infect people and many mammals.Human infection are primarily dueto eating raw or undercooked meat containing infective larvae,such as pork.With the change in eating habits, wildlife and horse become a new source ofinfection for human trichinosis.Many countries around the world havereported outbreaks of the disease, and the disease have a great impact onanimal breeding and food safety.Therefore, the development of a vaccinedefence against trichinosis will have great economic value.Commonly used methods to prevent parasite infection is the use of itsnatural antigen or recombinant protein antigen for immunization of thehost,then play a protective role.T. spiralis crude antigen preparations, ESproducts,and recombinant proteins-based vaccines have been reported usinganimal experiments. Such as p43, p53DNA vaccines, animal experimentsdemonstrated that the vaccine has immune protection for host. However, dueto the trichinella antigens changes during the development in the host and itscomposition is very complex,it is difficult to induce high levels of protectionwith developed vaccine，we should be actively looking for more effectiveantigen gene.From trichinella infective animal models,using the method of artificialdigestion collected muscle larvae,then using RT-PCR cloning of a new gene.The sequencing results compared with the published gene sequence,the resultsconfirmed that both come from the same gene.Recombinant proteinswasacquired using prokaryotic expression,protein purification,Western Blotmethods,the results confirmed that the antigenic protein for vaccine development has laid a theoretical foundation.Objective:Cloning and expression of a new Trichinella spiralis antigens thatMyophilin,and exploring it’s initial immunological characteristics.For theprevention of trichinosis provide the scientific foundation.Methods:Genome of Trichinella spiralis was acquired from the GenBank databaseand obtained the cDNA sequence of Myophilin,then designed the primers usethe Primer5.0softwar（eUpstream and downstream primers containing Nde Ⅰ,Xho Ⅰ restriction sites）.For extracting the total RNA,Trichinella spiralismuscle larvae were collected as a template for RT-PCR.The PCR productswere run on2%agarose gel, and the target gene bands plastic were cut byrecycling. The recovered DNA and cloning vector pMD-19T were digestedrespectively by the two kinds of enzymes Nde Ⅰ and Xho Ⅰ.Digestionproducts connected each other with3:1,and then transformed into the DH5aEscherichia coli competent cells,cultured by Amp resistance medium. Positiveclones were identified by PCR and double digestion, both positive ones weresequenced, then the results were compared with the nucleotide sequence fromthe GeneBank by the DNAMAN analysis software to analyze homology. Therecombinant pET22b-Myophilin was constructed then expressed in E.coli withinduction by IPTG,His-tagged affinity purification.The recombinant proteinwas identified by SDS-PAGE,then detected the protein of immunogenicity bywestern blot.Results:1. The Myophilin was cloned by RT-PCR: a579bp band was amplifiedby RT-PCR,consistent with the expected size,indicating that the Myophilinwas successfully amplified.2. The recombinant plasmid was identified by PCR and double digestion:the recombinant plasmid as a template,PCR amplification after agarose gelelectrophoresis to obtain a strip fragment size579bp.Recombinant plasmidwas digested by NdeIand XhoI,got the size of2600bp,579bp and300bp bands, consistented with the expected results, verified again the purpose of cloningsuccess.3. The Myophilin’s sequence detection and homology analysis:themeasured Myophilin sequence compared to the gene sequence from databaseNCBI,it homologous to100%.4. Constructed recombinant expression vectors and identification:thepET22b expression vector and objective gene both were digested by NdeI andXhoI, then connected with each other,abstracted plasmid from the connection,a579bp band was obtained by plasmid PCR,then digested to obtain the size of5500bp and579bp bands,the results proved a successful connection.5. Purification of recombinant proteins: the recombinant protein waspurified by6*His-tagged Ni-affinity chromatography,then got a21kDa band.6. Immunological properties were identified by Western Blot: the purifiedprotein by SDS-PAGE electrophoresis,the mice serum which infected withTrichinella for40days performed as an antibody for western blot.The21kDaband appeared,described Myophilin antigenic.Conclusions:1Successfully extracted the total RNA from Trichinella spiralis Musclelarve, cloned Myophilin gene.2Constructed prokaryotic expression vector of recombinant Myophilingene.3The Myophilin protein was induced expression and successfullypurified.4Used the Western Blot way to verify the Myophilin antigen’santigenicity.