Full Genomic Analysis Of Transmissible Gastroenteritis Virus(TGEV)TZ-10-2016 And Establishment Of Indirect ELISA Method

Transmissible gastroenteritis virus(TGEV)of swine is an acute,highly contagious infectious disease which has a worldwide prevalence and high mortality,and seriously endangers the pig industry.It is one of the important pathogens causing diarrhea in piglets.In this study,a porcine transmissible gastroenteritis virus was isolated and identified by RT-PCR and electron microscopy.Through cellular virus proliferation,Genome-wide sequence analysis,and the His-Sa soluble recombinant protein was constructed for the TGEV-Sa gene.The expression product was used as coating antigen to establish an ELISA for detection of TGEV antibodies.1 Isolation and identification of TGEV-TZ-10-2016Isolated from a diarrheal disease in a pig farm in Jiangsu Province and a suspected porcine transmissible gastroenteritis virus was obtained and named TGEV TZ-10-2016.A specific primer was designed for the TGEV-Sa gene and was amplified by RT-PCR to obtain a 507 bp target segment as expected.The sequencing results showed that the nucleotide homology of TGEV-TZ-10-2016 and the known TGEV strain is above 94.6%,High homology with Chinese TGEV strains(PURDUE P115,TGEV-HX,TGEV-SHXB,WH-1,AYU,SC-Y)and US strain(PUR46-MAD).Combined with electron microscopy observations,it was confirmed that the isolated virus was porcine transmissible gastroenteritis virus.2 Whole genome analysis of TGEV-TZ-10-2016This study was conducted to obtain relevant information on the whole gene sequence of the TGEV-TZ-10-2016 and found the clinical samples of the isolated TZ-10-2016 had cytopathic effects after 18 passages in pig testis cells.With reference to the published TGEV sequence in GenBank,18 pairs of specific primers were designed and synthesized,which were used for the whole genome amplification of TGEV-TZ-10-2016.The cDNA sequence was then spliced and the genetic variation and bioinformatics were analyzed.The results showed that the complete genotype of TGEV-TZ-10-2016 was 5'-ORFla-ORFlb-S-ORF3a-ORF3b-sM-M-N-ORF7-3'.The homology of TGEV-TZ-10-2016 were similar with TGEV-SHXB,WH-1,AYU,PUR46-MAD reached 99.5%;Bioinformatics analysis showed that the structural protein S,sM and M are all stable proteins and have transmembrane regions.N protein has a certain degree of hydrophobicity.All four proteins have good antigenicity.This study provides a reference for the study of porcine transmissible gastroenteritis virus.3 Establishment of Indirect ELISA methodThe target fragment was obtained by RT-PCR with the designed TGEV-Sa gene-specific primers and was cloned into the prokaryotic expression vector pCold-TF.After the double enzyme digestion and sequencing,the correct recombinant expression plasmid was transformed into BL21 and induced with a final concentration of 0.5 mM IPTG and most of the expressed products were soluble protein.After ultrasonication of the bacteria,the recombinant protein was purified by using a Ni-NTA kit.The results of Western-blot identification was correct,and the successful expression of the recombinant protein laid the foundation for the next step of the ELISA method.An ELISA for TGEV antibody detection was established by using the purified His-Sa recombinant protein as a coating antigen.Through the optimization of experimental conditions,it was confirmed that the optimal amount of antigen was 0.0625 ?g/mL,the best dilution of the tested serum was 1:100,the optimal incubation time of first antibody was 45 min,the incubation time of the enzyme-conjugated secondary antibody was 45 min,and the color development time of the substrate was 15 min,40 negative sera were tested with the established ELISA to determine the positive critical value and the result showed that when OD450>0.331,it was judged as positive.Cross-reactivity test found that the ELISA established in this study had good specificity that it responded to TGEV positive serum but not responded to Porcine rotavirus(PoRV)and Porcine epidemic diarrhea virus(PEDV)positive serum.The coefficient of variation of the intra-assay and inter-assay experiments of this method was less than 10%,which indicated that this method had good reproducibility.282 pig serum samples were detected with ELISA and indirect immunofluorescence and the results showed that there coincidence rate is 88.3%,which proved that ELISA established in this study can be used for clinical testing.