Cloning Of The Completed Genome Sequence Of Two Porcine Coronavirus And Defective Minigenome Construction And Expression Of Structure Genes

PEDV and TGEV are numbers of coronavirus genus which belongs to coronaviridae[0] family, and caused the diseases are characterized by severe enteritis and diarrhea. In recent years, the research about the PEDV and TGEV has been made great progess. In this study, based on the PEDV and TGEV samples, cloning the genome of its and construction the expression vector and derived synthetic minigenome, the results are as follows:1. Cloning and analyses of the structure and non-structure genes of PEDV DX strain. The results show that the S, M , sM and N genes open reading frame (ORF) were 4152, 681, 231 and 1326 bases long respectively, the replicase gene and ORF7 were 20354 and 675 nucleotides.The S gene ORF of of PEDV DX strain long as that of the CV777, JS-2004-2 and Chujin99, but shorter than that of strains which accession numbers were AF500215 and AF 237764. PEDV DX has a high degree of homology with the JS-2004-2strain.2. Construction the prokaryotic vector and expression of PEDV N gene. The result show recombinant protein possessed native biological activity.3. Construction the TGEV and PEDV S+N recombinant adenovirus expression vector respectively, green florescence show the recombinant vector can express foreign genes.4. Cloning and analyses of the genome of TGEV TS strain. The results show that the genome of TGEV TS was 28541 nucleotides, the S, M , sM and N genes were 4350, 786, 246 and 1146 bases long respectively; the replicase gene, ORF3 and ORF7 were 20054, 1015 and 237 nucleotides, the 5'and 3'UTR (un-translated region) were 319 and 280 nuleotides respectively.The replicase 1a gene was predicted to encode a protein of 4017 aa while the replicase 1b gene was predicted to encode a protein of 2680 aa. It was not 4017 aa and 2678 aa as reported. The phylogenetic trees revealed that TGEV strains compared were divided into 2 groups, and that the TGEV TS strain shared the closest relationship with Miller group.5. After passaging in swine testicle (ST) cells, Porcine Kidney (PK-15) cells and Porcine kidney cell line IB-RS-2 (IBRS) for 10 generations, cytopathic effects (CPE) were observed after 36 hours in ST cells and 3'genome could be cloned, CPE were observed after 48 hours in PK-15 and IBRS cells but the 3'genome could not be cloned. After 50 generations, 3'genome could not be cloned from infected IBRS cells, the 3'genome from ST and PK-15 cells indicated that more mutations had happened in PK-15 cultured than in ST cultured.6. Construction the TGEV TS defective minigenome (DI-M33), GFP gene was cloned into the DI-M33, green florescence could be observed in infented ST cell by the helper virus.