Clone And Expression Of NSP 2C Gene Of EMCV TJ Isolates And Development A Detection Method Of ELISA

Porcine encephalomyocarditis is an acute infectious disease caused by encephalomyocarditis virus (EMCV), characterized by encephalitis, myocarditis or inflammation around cardiac muscles in piglets, and reproductive failure in sows and gilts. It was a zoonosis,not only brought serious economic losses for pig feeds but to danger the human-beings health. The 2C gene participateed in the replication of EMCV and The 2C protein was a good immune antigen .In this study,the 2C gene was cloned,the 2C gene's prokaryotic expression vector pGEX-6P-2C of TJ strain was constructed,the 2C protein was gain and an indirect ELISA was established by using the purified EMCV recombinant protein 2C.The rearch has began to do for molecular biology of virus, molecular epidemiology and produce of vaccine in our country.At first, the encephalomyocarditis virus of TJ stains was inoculated onto Baby Hamster kidney cell line (BHK-21) and the result showed that the virus can coused cytopathogenic effects (CPE) after 24h.A special primers of the3D gene were designed to identify the fragments propagation of inoculated BHK-21 and the cDNA of of TJ isolates 3D gene were amplified by RT-PCR.The result showed that the lenth of 3D gene was 286 bp and the nucleotide and amino acid identity among others isolates was 100%.Second, According to reference strains in GenBank ,a special primers were designed to amplify the 2C gene by RT-PCR , the gene was successfully amplified and the length of it was 978 bp. The homology of the genomic nucleotide sequence were aligned and analyzed between TJ and other EMCV stains available in GenBank.the result showed that the 2C gene shared 80.8%～99.8% identidy with those of EMCV reference stains ,and showed the highest homology of genomic nucleotide sequence with BJC3 99.8%,the higher with HB1 99.7%,but a lowest homology with mengo virus,only 80.8%.The target fragments was cloned into prokaryotic expression vector pGEX-6P-1. Induction with 0.1mM IPTG ,the recombinant protein was highly expressed as the form of inclusion body in E coli. cell BL21 and the molecular weight of pGEX-6P-1 was 61.6 ku. After the expression proteins pGEX-6P-2C were purified by washing and SDS-PAGE, PVDF analysis with the porcine polyclonal antibodies against EMCV showed the purified recombinant protein 2C could be recognized by the positive serum from the pig infected with EMCV.At last, an indirect ELISA was established by using the purified EMCV recombinant protein 2C. The method was applied for detection of EMCV antibodies in 327 serum samples which was collected from from 91 the extensive swine farms in 7 regions of Tianjin in 2006.We found that the antibody of EMCV from 86 the extensive swine farms in 260 serum samples were positive .The Sero positive rate of EMCV from tested farms ranged from 75% to 100% and the average rate was 91.24% .The Sero positive rate of EMCV between 58.33% and 95.19% ,and the average rate was 83.18%.The result indicated that EMCV was popular on pig farms in Tianjin. Otherwise, 295 serum random samples from 327 were detected again by coating antigen pGEX-6P-VP1.The result showed that the otherness of Sero positive rate was not obvious between 2C and VP1 protein ,but the Sero positive rate detected by 2C protein was lower than by VP1 protein .