Expression Of B And C Antigenic Sites Of TGEV Spike Gene In Prokaryocyte And Preparation Of Nucleic Acid Probe At 5' Terminus

Transmissible gastroenteritis ( TGE ) was an enteric disease of pigs caused by transmissible gastroenteritis virus (TGEV), a member of coronaviridae family. TGEV was characterized with high mortality and had spread all over the world. The genome structure of TGEV, PEDV and PRCV had high homology and it was difficult to diagnose TGEV by traditional means.Two studies were carried out to differentiate the TGEV from PRCV and investigate the epidemiology of TGE. One was cloning, sequence analysis and expression of the fragment containing the B and C antigenic sites locating at the 5' terminus in spike gene of TGEV in prokaryotic expression system (fused with GST), the other was preparation of non-radioactive probe labeled by digoxigenin for detecting the RNA extracted from TGEV by assay of Dot-Blot.Spike protein was the main protective antigen inducing the neutralization antibody. The fragment containing antigenic sites B and C locating at the 5' terminus in spike gene of TGEV was deleted in PRCV. The 1st pair of primer was designed according to strain TH-98.The fragment (named ts) was amplified by RT-PCR and inserted into pMD18-T vector. Sequence analysis showed that homology of nucleotide and amino acid between ts and other TGEV strains Fs772/70, Miller, Purdue15, Pur46-Mad, 133 were 96.92%, 97.76%, 98.88%, 99.72%, 99.44% and 94.92%, 95.76%, 97.46%, 99.07%, 98.31% respectively. Fragment ts was subcloned into expression plasmid pGEX-6P-1, then was transformed into competent cell of BL21 (DE3) pLysS. After screening the positive colony containing recombinant plasmid pGEX-6P-ts, the E.coli was induced by 0.2mmol/L IPTG and was analyzed by SDS-PAGE. The overexpressed protein (TS) was 13.2 ku and showed 40ku in gel fused with GST (26ku) . The amount of TS expressed was 37.9% under optimized condition.The 285 bp fragment (named tp) , locating at 5'terminus of spike gene, had high conservation in TGEV isolations. The 2nd pair of primers was designed according to the sequence of strain TH-98 collected in GenBank. Concentration of tp was analyzed after amplification invitro by RT-PCR, purified by low melt agarose and labeled by digoxigenin. After the estimation of labeling efficiency tp was applicated to hybridize the RNA of TGEV, then was subjected to self-development of X-ray exposed by CSPD to show the target nucleic acid. The minimal amount of TGEV cDNA can be detected was about 120 pg in this research.In the research TS containing antigenic sites B and C was expressed with high efficiency. Nonradioactive nucleic probe to TGEV was prepared and was applicated to detect TGEV atfirst stage, which established foundational work for differentiating TGEV from PRCV and investigation on epidemiology of TGEV.Candidate: Hu Sen Major: Preventive Veterinary ScienceSupervisor: Prof. Wang Junwei...