CDNA Clone Of Transmissible Gastroenteritis Virus Genome And Expression Of The Major Antigens Of Spike Protein In Pseudorabies Virus

Transmissible gastroenteritis virus (TGEV) is a member of the coronaviridae, and causes gastroenteritis and severe diarrhea in pigs, resulting in high mortality in neonates, which is greatly harmful for the stock raising and suffers heavy losses in the worldwide development of stock raising. The genome of the TGEV is 28.5 kb in length. It is a single-stranded positive-sense RNA virus.The virion RNA functions as a mRNA and is infectious. About 20 kb of the 5'two-thirds of the entire RNA comprises open reading frames(ORF1) encoding the replicase polyprotein, and 8.3 kb of the 3' one-third of the genome includes the genes encoding structural(spike protein, envelope protein, membrane protein, nucleoprotein) and nonstructural proteins. All of these proteins are essential in the life cycle of the TGEV. Spike protein plays role in biological function in the life cycle of the virus: firstly, spike protein is the only stuctructional protein that contains antigenic determinant and can induces swine to generate antibody; secondly, spike protein contains the acceptor of aminopeptidase N, which decides the cell tropism of virus; thirdly, spike protein is one of the virulent genes of TGEV. fourthly, spike protein helps TGEV enter into cytoplasm from cytoblast. Up to now, the function of some genes of TGEV have still been not understood. In this article, the full-length cDNAs were amplified by RT-PCR, and some of them were cloned. The prepare for the research of gene function, pathogenesis and vaccine of TGEV in the future.According to the sequences of TGEV in GenBank, 6 pairs of primers were designed. TGEV-31 (1.5 kb),TGEV-32(2.0 kb),TGEV-33(1.1 kb),TGEV-34(1.4 kb),TGEV-35(1.4 kb),TGEV-36(1.3 kb) fragments were amplified by RT-PCR, and were cloned into pGM-T vector, designated pGMT-31, pGMT-32, pGMT-33, pGMT-34, pGMT-35, pGMT-36. The sequences were determined by TAKARA, which are 1456 bp,1957 bp,1079 bp,1362 bp,1427 bp,1327 bp in length, respectively. Bioinformatic softwares assemblyed all the 6 sequences to a 8495 bp sequence. According to the phylogenetic tree based on TGEV S,M,sM,N,Nsp3a,Nsp3b,Nsp7 genes, TGEV is close to the TS,HN2002,TO14,TS,PURDUE isolates of TGEV.According to the sequences of TGEV in GenBank, 7 pairs of primers were designed. TGEV-A(6.2 kb),TGEV-B(5.3 kb),TGEV-CI(mu)(2.3 kb),TGEV-C2(mu)(3.0 kb),TGEV-DE(6.9 kb),TGEV-F(5.1 kb) PCR fragments were amplified by long RT-PCR. Point mutation was applied for amplifying the full-length of TGEV-C(5.3 kb) TGEV-A, TGEV-B, TGEV-C, TGEV-DE, TGEV-F were ligated to pGM-T vector, but TGEV-A, TGEV-B, TGEV-C were only cloned to pGM-T vector successfully, designated pTA-A,pTA-B,pGM-T.According to the sequences of TGEV in GenBank, one pairs of primers were designed for amplifying the major antigens of spike gene, and the PCR fragments were cloned to the pET32a,pET28a and pEGFP-Cl vectors, designated pET32a-S, pET28a-S, pEGFPC1—S. Expression was carried on in Ecoli BL21 and Vero cells, resulting in the expression in Vero cell, but not in Ecoli BL21 cell.According to the sequences of TGEV in GenBank, one pairs of primers were designed to amplify the major antigens of spike gene, and the PCR fragmfent was cloned to the pPPI2.EGFP transfer vector which had been constructed previously of PRV, designated pPPI2.EGFP.S, pPPI2.EGFP.S was co-transfected with PRV SA215 DNA to the Vero cells by calcium acid phosphate, resulting in the fluorescence 12 h post-transfection and the highest about 24-48 h post-transfection, and begining to decline 72 h post-transfection. CPE begined to appear 48h post-transfection and about 75% cells appeared CPE. Recombination virus, designated PRV SA215-S, was constructed by purifying the cells which both appeared CPE and fluorescence. The recombination virus was identified by PCR, and PCR fragment 1.7 kb in length was amplified.Codon bias of TGEV and PRV were analysised by bioinformatics, and RSCU,Nc,GC,GC_3s and bi-nucleotide values were accounted to study the codon usage of PRV and TGEV. GC_3s-Nc plots ,GC₁₂-GC_3s plots, and correspondence analysis were applied to study the major factors of shaping the synonymous codon usage of PRV and TGEV. RSCU,Nc,GC,GC_3s and bi-nucleotide values reveal that PRV mainly biases at the codons ending with G and C, while TGEV biases at the codons ending with A and U. GC_3s-Nc plots, GC₁₂-GC_3s, plots, correspondence analysis reveal that compositional bias, mutational bias, translational selection, together with funcation of gene shape the synonymous codon usage of pseudorabies virus, while compositional bias, mutational bias are the main factors shaping the synonymous codon usage of TGEV.