Expression And Purification Of HCV-Core, NS5A Gene Fusion Protein

Objective Hepatitis C caused by the infection of Hepatitis C virus(HCV) is one of the main causes of chronic Hepatitis and the cause of hepatic cell cancer(HCC);however,there are lack of efficient therapies for HCV infection nowadays.Study the pathogenetic mechanism of HCV is of great importance,this text construct proeukaryotic cell expressive vector of HCV-Core and HCV-NS5A gene,abundantly express and purify.throgh this method to prepare their multiclone and monoclonal antibody,the purpose of the study is to clarify biological function of HCV Core and NS5A gene in the course of HCC through the following aspects,such as template PCR,Restriction analysis,transformation,induction,prokaryotic expression,protein purification.Methods(a)The DNA fragment of HCV-Core was amplified bypolymerase chainraction(PCR) by using DNA from plasmid of as template.After sequencing,the correct DNA fragment was inserted into inducible proeukaryotic expressive vector pET-32a(+) and transformed into the competent E.coli BL21.E.coli was induced with IPTG,and cell iysate was analyzed with SDS-PAGE and conformed with Western blot.Expressed bacteria were quassationed by supersound and analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The expressed product was purified and renatured by Ni+ Affinitycolumn chromatography.(b) The DNA fragment of HCV-NS5A was amplified bypolymerase chainraction(PCR) by using DNA from plasmid of as template,the DNA fragment was transformed into the competent E.coli DH5α.Extract plasmid,After sequencing,the correct DNA fragment was inserted into inducible proeukaryotic expressive vector pET-32a(+) and transformed into the competent E.coli BL21.E.coli was induced with IPTG,and cell lysate was analyzed with SDS-PAGE and conformed with Western blot.Expressed bacteria were quassationed by supersound and analyzed with sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE).The expressed product was purified and renatured by Ni+ Affinitycolumn chromatography.Results(a) The Core and NS5A fusion protein was highly expressed in the prokaryote expressing system.(b)The protein production mainly was in inclusion body through SDS-PAGE analysis.(c) Through the expressed product was purified and renatured by Ni+ Affinitycolumn chromatography.The purified proteinCore and NS5A was obtainedConclusions The Core and NS5A fusion protein was induced and expressed successfully by E. coli prokaryote expressing system.The expressed protein was purified by affinity column chromatography and highly specific and potent polyclonal antibody of Core and NS5A was prepared.The above results provided an valuable information for the further study on the meaning of Core and NSSA gene in the course of HCC by other methods such as immune histochemistry,, cellular proliferation and signal transduction.