Cloning Of Genome Of Porcine Transmissible Gastroenteritis Virus And Expression Of N Protein

The mortality may reach 100% for pigs under 2 week old infected bytransmissible gastroenteritis virus. Geat loss for worldwide development of stockraising has been generated from prevalence of transmissible gastroenteritis(TGE).TGEV belongs to the family coronaviridae. The genome of the TGEV is 28.5 kb inlength. It is a single-stranded positive-sense RNA virus.The virion RNA functions asa mRNA and is infectious. About 20 kb of the 5' two-thirds of the entire RNAcomprises open reading frames(ORF1) encoding the replicase Polyprotein, 8.3 kb ofthe 3′one-third of the genome includes the genes encoding structural andnonstructural proteins, all of these proteins are essential in the life cycle of the TGEV.Understanding the genome and exploring the function of the essential proteins ofthe isolated strains of TGEV may aid us in elucidation the molecular geneticspathogenesis of the virus, and provide the important bases for research of TGEVmolecular biologies, diagnostics and genomics. In this study, on the basis of biologycharacteristic of TGEV,one strain of TGEV, designate as SC-Y, was successfullyisolated from diarrheal piglet fecal samples from sichuan province. Sixteen cDNAfragments covering the whole genome of the SC-Y strain of TGEV were obtained.The expression of the important structure protein N was studied.The possible positive samples were homogenate and the supematant wereinoculated to the ST cells, after eight passages, the isolate could produce obviouscytopahic effects(CPE). The TGEV replicated on ST cells were examined through thedirect fluorescent antibody test, the specific immunofluorescence was watched mostlyin the cytoplasma. The cells inoculated SC-Y were harvested to make section forTEM examination, typical eoronavirus particles were seen in the endoeytoplasmicreticulum. A pair of primers was designed based on the published N sequence of TO14, Purdue, Purdue-115 and TS strains. The expected DNA fragments wereamplifed by RT-PCR from the infected ST cells, and were cloned into PMD18-Tvector and further sequenced. The results of sequencing showed that SC-Y isolategene N is 1149 bp. Homology of the N gene is more than 95.0% compared with thepublished strains in the GenBand.Sixteen pairs of primers coveting the whole genome of TGEV were devisedaccording to the published sequences as well as analysis of the restrictionribonuclease map ,16 cDNA fragments were amplified by RT-PCR. The amplifiedfragments were further cloned and sequenced. The size of complete genome is 28,590nucleotides (including Poly A), and comprises of 7 ORFs, which is flanked byuntranslated regions(UTRs), 315 bases at the 5′-end and 277 bases at the 3′-end.Comparison with other isolates, SC-Y is found to very high homology withPUR46-MAD strain. Phylogenetic analysis of TGEV suggested that SC-Y may belongto same genogroup with purdue strain.3 cDNA fragments about 5 kb were obtained by LA-PCR amplifications andgradual ligations between overlapping 8 cDNA fragments of recombinant plasmid. 2cDNA fragments about 6kb were amplifed by RT-PCR from the infected ST cells.These cDNA covering the complete genome and overlapping fragments A,B,C,D,Ewith a size of 6228 bp,5247 bp,5275 bp,6911 bp and 5115 bp were cloned into PTA2vector,which provided the conditions for the construction of the full-length TGEVcDNA Clone.TGEV SC-Y has slightly bias at the codon which are T and A at the end.Yeast system should be more suitable for TGEV' gene expression. Sequencecomparison showed that the ORF3a and 0RF3b of SC-Y share low homology withother isolates., the ORF1b is highly conserved compared with the publishedstrains. N protein was predicted to be comparatively conservative protein.The secondary structure of 5′UTR and 3′UTR showed some differences existamong different isolates.The recombinant plasmid pMD18-N was digested with Bsp1191 and XhoⅠtogeneraet N gene,and subcloned into the prokaryotic expression vector PET-32a(+). The recombinant plasmid was transformed into BL21. The recombinant bacteria wasinduced by IPTG. The expression of N protein was optimized with proper inducingconditions of 0.2 mmol/L IPTG, 4 hours and 25℃temperature. Using the puridiedfusion protein as coating antigen, the optimal concentration of N for coating of platewas 15μg/ml; the dilution of antibody was 1:40; the best blocking solution was0.5%FBS; serum sample for detection should be incubated for 90 min; the workingconcentration of HRP-SPA was 1:5000, HRP-SPA should be incubated for 60 min; thesubstrate for ELISA was incubated at RT for 10 min. Following the determination ofcondition of ELISA, the threshold value of ELISA was 0.35. An indirectenzyme-linked immunosorbent assay(ELISA) based on the N protein was developed.