Construction Of Porcine DC-SIGN Eukaryotic Expression Vector And Preliminary Validation Of The Role Of DC-SIGN In The Process Of PEDV Infection

Porcine epidemic diarrhea(porcine epidemic diarrhea, PED) is caused by porcine epidemic diarrhea virus(pig epidemic diarrhea virus, porcine epidemic diarrhea virus) by diarrhea, vomiting, dehydration as the main characteristics of an intestinal infectious diseases, on piglet death rate up to 100%. Since the end of 2010, acute PED outbreaks have continually occurred in most provinces and cities in China, which have resulted in substantial economic losses. Therefore, basic research regarding the pathogenesis of PEDV has important significance. C-type lectin DC-SIGN specificexpression of a multifunctional protein molecules on dendritic cell surface and infection in the PEDV belong to the SARS coronavirus(SARS CoV), feline infectious peritonitis virus(FIPV), avian infectious bronchitis virus(IBV) and human coronavirus 229E(HCoV-229E) play an important role. This shows that the role of DC-SIGN in PEDV infection in pigs, to provide basic information for the analysis of the mechanism of PEDV infection.In order to obtain porcine DC-SIGN polyclonal antibody. This study according to the GenBank: DC-SIGN nucleotide sequence(EU684956). In E.coli codon preference, the artificial synthesis of the pig DC-SIGN gene(pDC-SIGN), and the 5’end and the 3’ end were introduced BamHI and XhoI cut points. pDC-SIGN gene was cloned into prokaryotic expression vector pGEX-6p-1, and the gene was successfully expressed in E.coliBL21. pDC-SIGN recombinant protein polyclonal antibody was prepared by immunization of BALB/c mice with purified pDC-SIGN recombinant protein. Western blot results showed that pDC-SIGN recombinant protein polyclonal antibody to identification of porcine intestinal epithelial cells(IEC), swine testicle cells(ST), porcine kidney cells(PK15) and small intestine tissue in natural porcine DC-SIGN and can be used for the detection of subsequent pDC-SIGN. In order to construct the eukaryotic expression vector of pDC-SIGN, the pDC-SIGN gene(pSIGN) was synthesized according to the preference of the eukaryotic host cell codon, and the EcoRI and XhoI were introduced at the 5’and 3’ ends of the gene. Immunofluorescence assay and Western blotassay demonstrated that the synthesized pDC-SIGN gene was transiently transfected into PEDV cells of non sensitive cells in the non sensitive cells of Chinese hamster ovary(CHO). Virus infected cell count experiments show that the expression of CHO pSIGN cells, PEDV infection in 12 h, 24 h, 36 h and 48 h, the number of infected cells was significantly higher than that of the control group(p<0.05). Of the research by the polyclonal antibody of pSIGN, constructed pSIGN eukaryotic expression vector and preliminary validation pSIGN in CHO cells in over expression can increase the cell number of PEDV infection and provide the material basis for pDC-SIGN the PEDV infection in the role of deep research.