Cloning, Sequencing And Expressing Of Protective Antigen Gene Of The Spike Glycoprotein Of Transmissible Gastroenteritis Virus

Transmissible gastroenteritis virus(TGEV) is the etiological of transmissible gastroenteritis(TGE),which is a highly contagious enteric disease in piglets causing vomi-ting,severe yellowish diarrhes, weight loss, dehydration, high mortality and resulting in severe economical losses to the swine farms.The spike(S) glycoprotein of transmissible gastroenteritis virus is the predominant inducer of neutralization antibody. It has four major antigenic sites(A,B,C,D).The sites A and D are known to be regions inducing major neutralization antibodies. So, cloning and expression of the antigen site A, the D is especially important.In this study, one pair of primers are designed and synthesized according to the published sequence of TGEV TH-98 strain S gene with Primer 5.0 software. S1 gene(A and D) was amplified by reverse transcriptase PCR(RT-PCR) from TGEV Jilin strain which was isolated with PK-15 cell line before and had been characterized. The product of RT-PCR was cloned into the pMD18-T vector. Identification of restrictionenzyme, PCR and sequencing indicated that the S₁ gene has been cloned successfully. S₁ gene was inserted into EcoRI and SalI multiple cloning sites of the expression vector pET-32a, which was named pET-S₁. The recombinant pET-S₁ plasmid was transformed into BL21 host cell of E.coli and induced to express foreign gene by IPTG with a finial concentration of 1mmol/L. SDS-PAGE analysis showed that the recombinant protein had been expressed, it was about 44KD.The protein could react with polyclonal antibody against TGEV by Western-Blot test. The result declared that the expressing protein shared a good antigencity.We can predicted that S1 gene can be also expressed in Lactic acid bacteria.It was prepared for establishing the expression vector of Lactic acid bacteria and developing genetically engineering vaccine of Lactic acid bacteria.It was also provided a foundation to diagnose the TGE.