Establelishment Of Real-time PCR For Detection Of Transmissible Gastroenteritis Virus And Subcellular Localization Of ORF7and Its Effect On The Virus Proliferation

Porcine transmissible gastroenteritis （TGE）， which caused by transmissiblegastroenteritis virus, is a highly contagious and urgent infectious disease. TGE, whichcharacterized by severe diarrhea, vomiting as well as dehydration, was classified as anotifible disease by OIE. Since the highly genetic recombination exists in TGEV, and itspathogenesis is poorly understood, combined with the co-infection and secondary infectionbetween TGEV and other virus and bacteria, it is very difficult for making progresss in thediagnosis and preparation of the vaccines. ORF7encodes a non-structure protein of TGEV.Available information indicates that, ORF7counteracts host defenses and modulates virusvirulence. But it is still unknown of subcellular localization and the mechanism of ORF7modulates virus proliferation as well as virulence. We established the Real-time PCR fordetecting TGEV, and then investgated subcelluar localization of protein ORF7as well as theimpact of over-expressed ORF7on virus proliferation and replication. From the experiments,following results were obtained:（1）N gene was amplified from TGEV by RT-PCR and ligated to cloning vetorpEASY-T1to construct correct standard plasmids. Then the standard curve and the Real-timePCR assay for detecting TGEV were developed successfully by optimizing the varity ofconditions. Data showed high specificity and good repeatability, and demonstrated that themethod was of high repeatability, which was100times higher than general PCR, and thelinear range was from1.0×101to1.0×107copies/μL.（2）Recombinant plasmids GFP-ORF7and ORF7-GFP were constructed by ligating theamplified ORF7into the vectors GFP-C1and GFP-N1. After double enzyme digestion andsequencing, the recombinant plasmids were transfected into ST cells. About234bp fragmentswas obtained by RT-PCR and approximately9ku protein bands was obtained by Western blotanalysis. These data were consistant with expected results and suggested ORF7expressedstablely in ST cells.（3）Subcellular localization and a singal peptide prediction were explorated afterrecombinant plasmids transfected into ST cells. Subcellular localization results suggested that the difference of fusion site between GFP and protein ORF7affected co-localization of thelatter, and a singal peptide existed in1～30amino acid of-NH2terminal of protein ORF7.（4）ST cells which have been transfected recombinant plasmids, were inoculated withTGEV. After infection34h, N gene and virus contents were detected by Real-time. Real-timeshowed over-expressed ORF7upgulated the expression of N gene and leads the reduction ofvirus content.The Real-time PCR assay for detecting TGEV has high sensitivity and specificity, whichprovided technical support for the diagnosis, prevention and cure of TGE. Through theresearch of ORF7, our study first showed the locating features of protein ORF7,over-expression of ORF7upgulated the expression of N gene and leads the reduction of viruscontent. These results provided new references for follow-up studies about TGEV ORF7.