Isolation And Identification Of Porcine Gastroenteritis Virus And Cloning, Sequence Analycis And Prokaryoticexpression Of The S Gene

Porcine transmissible gastroenteritis (TGE) is caused by porcine transmissible gastroenteritis virus(TGEV) and affects all kinds and ages of pigs leading to enormous losses to the pig industry .In this study, a pair of primers were designed based on the sequence of the TGEV genomes (NC002306),and the RT-PCR to detect TGEV was developed. The 497bp specifical fragment was obtained from the TGEV vaccine strain RNA by this RT-PCR while it was not obtained from the PCV2, PRV, PRRSV and HCLV. The sensitivity of RT-PCR reached to 20 pg of detection for TGEV-S-cDNA.The pathogenic detection of TGEV in clinic suspected samples collected from XinxiangYuanyang(XXYY),Kaifeng(KF),Xinzheng(XZ), Zhu madian (ZMD), Xinxiang HuoJ-ia(XXHJ), Nanyang(NY)of Henan province were carried out by the RT- PCR . The 497bp specifical fragments were obtained from each sample above,and it could be digested into two segments of 373bp and 124 bp by Banll.The result was consistent with the TGE Ag rapid test kit . These results indicated that the RT-PCR was more specific and sensitive to detect TGEV and could be applied to do TGEV diagnoses and epidemiology investigation.Another pair of primers were designed and synthesized based on the sequences of the TGEV genome(NC002306),and S gene was amplified by RT-PCR from 5 cultures of PK-15 cells inoculated with TGEV samples and cloned into pMD18-T vector or PGM-T vector.The recombined plasmids PGM-XZ-S, PGM-NY-S, PGM-YM-S, PMD-XX-S and PMD-KF-S were obtained. Analysis of S gene shown that it was cloned successfully.Sequence and phylogenetic analysis confirmed that the nucleotide homogeneity for Henan strains is very high , they shared 99.0%-99.8% and 97.0%-99.0% homologies at their nucleotide and amineacid level respectively.and shared 99.0%-99.7% and 97.0%-98.5% homologies at nucleotide and aminoacid level with YM strain. It was found that strain XZ displayed the highest nucleotide and amine acid homology with strain Purdue(99.7% and 99.0%)train XZ has the highest amine acid homology with SC strain (100%), while strains XX, KF and NY shared 99.0%,98.5% and 99.5% amineacid homology with strain SC. It is suggests that S gene of different strains of TGEV is extremely conservative,though different isolates vary in their genomic sequences.A fragment of 592bp of S gene was amplified by PCR from the recombined plasmid PGM-XZ-S and cloned into pET32a(+) expression vector.Then the pET-XZ-S recombination plasmid that contains the S gene was obtained. Identification of pET-XZ-S was conducted by restriction analysis and nucleotide sequencing. pET-XZ-S was transformed into Escherichia coli BL21(DE3) to express, the optimal inducing concentration of IPTG was 0. 6mmol/L, and the optimal inducing time was 3 h.The molecular weight of fusion protein was 42KD. Western-blotting showed that fusion protein was able to specifically react with pig serum against TGEV. The expressed protein could be used as subunit vaccine, diagnostic reagent and so on.