Detection Of The Two Porcine Coronavirus And Cloning And Sequence Analysis Of Major Structural Genes Of Porcine Enteric Coronaviruses Isolated From China Recently

A multiplex reverse transcriptase PCR (RT-PCR) procedure was developed for the simultaneous detection of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) in pigs. The membrane gene of PEDV and spike gene of TGEV were chosen as targets. The PCR products of PEDV and TGEV had molecular sizes of 467bp and 1062bp, respectively. Primers from PEDV did not react with any TGEV and other porcine viruses. The multiplex RT-PCR was able to detect lpg tissue culture-infective doses of PEDV or lpg TGEV with each of the primer sets for PEDV and TGEV, respectively. The RNA of PEDV and TGEV were detected directly in intestinal and faecal samples from pigs infected experimentally with either virus. The detect result of 158 shares of clinical samples showed: PEDV and TGEV are ubiquitous in China and PEDV is more severe (53.2%). Duple infection of PEDV and TGEV is only 4.4%.The Strains named as PEDV JS-2004-2, PEDV AH-2004 and TGEV HN2002, TGEV JS-2004 were isolated in China recently. Twelve pairs of primers were designed to amplify S, M and N gene. The RT-PCR products were cloned into lined pMD18-T plasmids respectively and sequenced with other homologous PEDV and TGEV strains. The results affirmed that HN2002 and JS-2004 strain shared more with TS strain. The S, M and N protein amino acid sequence homology of JS-2004-2, AH-2004 and CV777 were more than95%. The PEDV JS-2004-2 and AH-2004 shared more.The viral subgenome mRNA of porcine transmissible gastroenteritis Virus (TGEV) was amplified by RT-PCR using primer pairs TS 1 and TS2 which were designed according to the reported reference sequence. A DNA fragment was amplified which contains segmentclosed with N terminal of S gene, and the size is about 922bp.The DNA fragment was cloned site specially into PET-32a vector between the EcoR V and Sal I cleavage site. The Pet-32a recombinanted plasmid was verified by restriction endonuclease analysis and nucleotide sequencing. Then it was transformed into E.coli strain Rosetta for S gene expression. The result of SDS-PAGE indicated that the production of recombinant TGEVS reached its peak about 5-8 hours after inducing at 28℃ and the expression protein can take about 25% of the total protein. The expression product was identified by Western blot test. A fusion protein about53.5kD as we expected was found. The results show that the vitro expressed protein of S gene via recombinant plasmid vector in the E.coli maintains antigenicity of TGEV.The recombinant TGEV S protein which was purified and the purified TGEV were used as based-antigen to set up two kinds of inderect ELISA.The two methods can distinguish TGEV serum which were negtive or positive obviously. The detection result of serum samples of pigs which time of life were 10d tol80d shows the better period of immunity is from 25d to 40d.