Cloning Of CD81 Gene And Construction Of Eukaryotic Expression Vectors Of CD81 Gene And Its Expression In Eukaryotic Cells

It has been estimated that approximately 3% of the world's population is infected with HCV. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide. The progression of prevention and therapy of HCV infection was hampered by lacking reliable cell cultures in vitro and successful small animal models.The pathogenesis of HCV is still not completely elucidated. HCV is a positive strand RNA virus of the flaviviridae family. Glycoprotein El, E2 on HCV envelope structurally form HCV highly ordered surface structure, where there are maybe ligand epitopes which combined with host cellular receptors. One of the first steps in a viral infection and propagation is the binding of the virus to cell surface molecules. It is one of the main factors that determining virus host specificity, tissue-tropism and pathogenicity.Based on Pileri's observation that by using the genomic library obtained from lymphocyte-derived cell lines, E2 protein of HCV specifically binds to CD81, this molecule is thought to present a putative cell surface receptor for HCV. The studies about HCVreceptor may help to explain their unusual host-scope, tissue-tropism and pathogenicity. Hence, blocking cellular entry of HCV by simulated moleculars assembling HCV receptor or receptor's ligand may provide new strategy for antivirus treatment.At present, there is not whole gene of CD81 ourselves. To understand the cellular entry and pathogenicity of HCV by simulated moleculars assembling HCV receptor, identify the expression situation of different cell lines for CD81, and reconstruct the cell model of receptor-mediated,we have done the following work:1. Cloning of CD81 gene ORFWe designed a pair of primers based on human CD81cDNA sequence publicity (NO:NM- 004356). Total RNA was extracted from the human Molt-4 cells as template according to the instructions and then human CD81 gene was was amplified by RT-PCR and PCR.The PCR products was determined by Agarose gel electrophoresis.2. .Construction of recombinant eukaryotic expression vectorsThe CD81 gene segement was cloned into pMD18-T vector by TA clone .The inserted gene sequence was anaylised. CD81 gene from the pMD18-T-CD81 vector with double-enzyme digestion was cloned into the pVAXl and pcDNA3.1(+) eukaryotic expression vector, named pVAXl-CD81and pcDNA3.1-CD81 correspondingly. The recombinant eukaryotic expression vectors pVAXl-CD81 and pcDNA3.1-CD81 were identified by PCR and restriction enzyme analysis in order to understand its right.3. Expression of CD81 gene in different eukaryotic cell linesThe recombinant vector pVAXl-CD81 and pcDNA3.1-CD81 were transfected into Africa green monkey kidney COS-7 cell line and human hepatocellular carcinoma HepG2 cell line respectively. And the transient expression products in the transfected cells were detected byimmunofluorescence assay (IFA), immunocytochemical staining andFACS.Result1. The CD81 gene was cloned successfully.2. The identification of the eukaryotic expression vector pVAXl-CD81and pcDNA3.1-CD81 by PCR and restriction enzyme analysis showed that CD81 gene was rightly inserted into the vector. Since the product of PCR and restriction enzyme analysis was 729bp approximately.3. The eukaryotic expression vector with CD81 gene was efficiently expressed on COS-7 cells.4. The human CD81 gene was successfully expressed in HepG2 cells.The transient expression product in the transfected cells was detected by FACS. Its fluorescence intensity (38.8%) was higher than the controls (19.6% and 1.4%) .Conclusion:l.The results showed that the CD81 gene could be cloned successfully by RT-PCR from human Molt-4 cells,and be used for research of receptor reconstruction.2. The heterogeneity COS-7 cells and human HepG2 cells are found negative for human CD81 expression,therefore, both heterogeneity COS-7 cells and human HepG2 cells are suitable for rebuilding of receptors and candidate cellular model of HCV infecti...