Development Of TaqMan Fluorescence Quantitative RT-PCR For Detecting Porcine Transmissible Gastroenteritis Virus And Prokaryotic Expression Of The Antigen Sites Of S Protein

Porcine transmissible gastroenteritis virus(TGEV) is described as causative agents of transmissible gastroenteritis of swine, causing piglets vomit, diarrhea and high mortality. It is a world disease and causes great harm to animal husbandry. Because of its high mortality, it is very important to detect the virus as soon as possible. So that, it is very necessary to constrcuct a quick and sensitive detection method. Traditional methods can not satisfy the requirement until fluorescence quantitative polymerase chain reaction(PCR) appear.In the study, the primers and probes were designed and synthesized according to the sequences of porcine transmissible gastroenteritis virus andβ-actin.The genes were amplified by reverse transcription polymerase chain reaction(RT-PCR) and cloned to the pMD18-T vector.Then we got the recombined plasmid, quantitated and ten-fold diluted. They were the standards. The reaction parameters were optimized to develop TaqMan fluorescence quantitative RT-PCR assay. Meanwhile, 60 field samples were detected and the results were compared with that of routine RT-PCR and TGEV antigen rapid test kit (chromatographic immunoassay). It was showed that the fluorescence quantitative RT-PCR assay could detect 15copies/μl of plasmid DNA and its specificity and reproducibility were very good, while the sensitivity of the routine RT-PCR was 1.53×10~3copies/μl. The result of field test also showed that its sensitivity was higher than that of the routine RT-PCR and TGEV antigen rapid test kit.Porcine respiratory coronavirus(PRCV) is the TGEV deletion mutant, and their homoly of nucleotide sequence reachs 96%. Because their genome structure is greatly similar and there is cross reaction between the neutralizing antibody, so that it is difficult to differentiate them by serological assays. Compared to TGEV, there are 621 - 681 nucleotides deletion at the N-terminal of S gene of PRCV, making PRCV lose two antigen sites.In this study, the S gene sequences from GenBank (ID:DQ811787) and our laboratory were aligned, and S gene deletion region of PRCV which was the B and C antigen sites(nt 91-513) of TGEV was amplified. Then the gene was cloned into pET30a(+) vector to express the protein. The result of SDS-PAGE assay showed that the protein was largely expressed and was about 45.4% of the total protein. The result of western blot assay showed that the protein has good biological activity. In this study, the protein was expressed and it is an important foundation to differentiate TGEV and PRCV by serological assay or preparing specific monoclonal antibody.