Random Panning Human CD81 Mimic Peptide With Phage Displayed Peptide Library

HCV Hepatitis C virus (HCV) is a major causative agent of acute and chronic hepatitis. It is estimated that there are 170 million HCV infected individuals worldwide. Infection is associated with the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma.and so far, interferon a combined with Ribavirin treatment is a commonly accepted therapeutic strategy. However, only about 40% of treated patients develop long-term responses.The HCV genome is a linear, positive-sense single-stranded RNA molecular of 9.5kb. It encodes a poly-protein precursor of about 3,000 amino acids. This poly-protein is cleaved by both host and viral proteases to generate several distinct polypeptides, with structural proteins located in the N-terminal portion and nonstructural protein in the C-terminal portion. From N- to C-terminus of the poly-protein, the component proteins are core protein(C), envelope glycoprotein (E1,E2) , and non-structural proteins P7, NS2, NS3, NS4A, NS4B, NS5A and NS5B.HCV E2 gp is distributed on the surface of viral particle. It may correlate with the immune response of virus and host cell.so it is the main role of preventing HCV infection.Truncated versions of E2 gp can bind specifically to human cells and were used to identify interaction with a number of cell surface molecules.among, human CD81 is accepted as the importment acceptor.though the mechanisms by which HCV enter target cells and CD81 mediate HCV infection are currently unknow. In vitro tests show CD81 can interact with soluble HCV E2 gp and virus in serum and is necessary for HCV entry. And the large extracellular loop(LEL) of CD81was found to be a critical determinant for viral entry, it is prospect to pan the mimic hCD81 from phage library to prevent HCV infection.In this study, we successfully expressed HCV E2 gp in CHO cells and human CD81 large extracellular loop(LEL) in E.coli;we screened hCD81 mimic peptide from 12 phage libray peptide library by using hCD81 mAb. suppression experiment showed the selected peptides could mimic the active of hCD81.1 ) HCV E2 gp expression in Chinese hamster ovary (CHO) cells and human CD81 LEL expression in E.coli.Using CHO cells as host cells,we transfected the cells with expression plasmid pCIDA-neo-E2.The primers were synthesized according to HCV lb genotype E2 gp nucleotide sequences to amplify the E2 outer segment genes . then neo genes were inserted among the eukaryotic expression plasmid pCI-neo intron. The 3' of neo genes was followed by E2 gp genes. That was the newly-constructed mammalian cell expression plasmid pCIDA-neo-E2. expression level was enhanced by eucaryon promotor. while the marked genes(neo) were lowly expressed with the help of intron.neo is the marked genes of G418,so we may screen stablely transfected cell clones. To constructe CHO host cells which could highly and stably express E2 gp, neo genes were insert among human globin introns, the introns would not infect the expression of objective genes as they were cut after mRNA transcription. When G418 among certain concentration were used to screen the cells, the concentration among which the cells could resist G418 had direct ratio with the expression level of neo gene. Hence,through screening, only the cells encoded highly integrated plasmid could survival, and the survival could highly express E2 gp.The cell culture was concentrated and conducted western bolt.different molecular weight of E2 gp were observed in cell lysate, and which owned the highest molecular weight was fully glycosylated E2 protein, the else were partly glycosylated E2 protein. We also detected the culture supernatant with dot blot and knew E2 gp could be secreted in the culture supernatant too.We also amplified hCD81 LEL cDNA,using plasmid pMD18-hCD81 as template, and inserted the product into prokaryotic expression vector pET32a.The recombinant plasmid pET32a-CD81 LEL encoded thioredoxin and human CD81 large extracellular loop. After induced by IPTG, the recombinant bacterium expressed dissoluble recombinant of about 28kD,which is similar with the molecular weight of interest protein.western bolt analysis showed that the fusion protein possessed the antigenicity of hCD81 LEL. ELISA detection showed that HCV E2 protein expressed in CHO cells could bind hCD81 LEL expressed in E.coli.2) panning with the ph.D. peptide library and detecting the activity of the productpanning was carried out by incubating a library of phage-displayed peptides with a plate coated with hCD81 mAb,washing away the unbound phage,and eluting the spedifically-bound phage.The eluted phage was then amplified and taken through additional binding/amplification cycles to enrich the pool in favor of binding sequences.After 3 rounds, 32 individual clones were characterized by ELISA and 3 high affinity clones were characterized by DNA sequencing at last. All the sequences conformed with those of ph.D. 12? peptide library. Their primary stucture shared no homology with those hCD81 which data were showed in GenBank. We could conclude that all of three peptides mimic the activity of hCD81.we coated the plate with HCV E2 protein to test if the phage clones could affect the binding of HCV E2 and hCD81. the results were consistent with what we had expected.Summary1 In this study,eukaryotic expression plasmid pCIDA-neo-E2 containing HCV E2 gp were expressed in CH0-K1 cell .Western blot and dot blot analysis showed the plasmids could express in the cells;prokaryotic expression vector pET32a containing hCD81LEL were expressed in E.coli. ELISA detection showed that HCV E2 protein expressed in CHO cells could bind hCD81 LEL expressed in E.coli.2 In this study,hCD81 mimic peptide were panned with the ph.D. peptide library using hCD81 mAb,ELISA detection showed HCV E2 gp could bind the phage clones preferly.