Prepare Monoclonal Antibody Of HCV Core Protein And Interreaction Of Core Protein And Apolipoprotein AⅠ And Translin

Objecive:To construct the prokaryotic expression plasmid expressing hepatitis C virus core antigen(HCVcore-Ag) and prepare monoclonal antibodies. To study interaction of HCV core protein and apoAⅠand Translin,and investigate the mechanism of this protein in fatty liver and cancer.Methods:DNA fragment cloing HCVcore-Ag was cloned into pET-32a ( + ) vecror using gene engineering technique. Recombinant plasmid was transformed into the competent E.coli BL21 and then proein was induced. Recombinant protein was purified by Ni-NTApurification column and acrylamide gel electrophoresis. Polycloal antibodied were developed by immunizing BALB/c mouse with purified rombinant protein,and the sensitivity and specifieity were tested by ELISA,Western blotting assays and immunohistchemistry. Constructed plasmid pDNA3.1-myc-his-apoAⅠ, pDNA3.1-myc-his-Translin,pDNA3.1(-)core,pact-apoAⅠ, pact-Translin and pBIND-core ,it were cotransfered into HepG2 cells.By using the methods of co-immunoprecipitation and mammalian two-hybrid system can detect interaction of HCV core protein and apoA1 and Translin.Results:1 : Recombinant plasmied expressing HCVcore-Ag was contracted successfully.A protein with the apparent molecular weight of 42kD was successfully expressing and purified.BALB/c mouse was routine immuned by purified protein,and was got blood from fromcaudal vein. Using ELISA to detecte antibody ,we foud that serum antibody titer was up-regulation.By Western blot we foud that core protein was detected in HCV liver cirrhosis patient using serum antibody titer.This shows that the structure is basi-conformity of fusion core protein and natural core protein.Cellular fusion of splenic cell and myeloma cell,the rate was 59.2%.By ELISA and Westren blot,we got 36 hybridoma cell.Using limiting dilution,we got 6 hybridoma cell which can stably secrete antibody.Using cell culture fluid of 4E10 hybridoma cell as antibody,by Western blotting sensitivity of limit is 8 ng;by immunohistochemistry,we foud that HCVcore protein extensively expressed cytoplasm of hepatic cell in HCV liver cancer.2: pDNA3.1-myc-his apoA1 and pDNA3.1(-)HCVcore were cotranstected to HepG2 cell.By CO-IP and Westren blot assay,the results show:zone of alone core protein was about 19KD,and zone of coprecipitation was 50KD. pDNA3.1-myc-his apoA1 and pDNA3.1(-)HCVcore can overexpressed in HepG2 cell,and both donnot be interacteded with other protein in HepG2 cell.Group of cell cotranstected pACT-apoA1 and pBIND-core was 12～16 times more than increase of luciferase level relative to the negative control.This show HCVcore and apoA1 can interacte in vivo and alonelly transtection have not autocatalysis.3: pDNA3.1-myc-his Translin and pDNA3.1(-)HCVcore were cotranstected to HepG2 cell.By CO-IP and Westren blot assay,the results show:zone of alone core protein was about 19KD,and zone of coprecipitation has only Translin protein, 21.8KD. Group of cell cotranstected pACT-Translin and pBIND-core was 6～8 times more than increase of luciferase level relative to the negative control.This show HCVcore and Translin can interacte in vivo.Conclusion:The recombinant HCVcore-Ag obtained in this study was highly purified and had a strong antigenicity.The polyclonal antibody developed had both high sensitivity and high specificity.This study provides new method for studyying biological function of HCV core gene. HCV core protein and apoA1 can interacte in vivo.This study provides new theory for the mechanism of chronicity hepatitis C in fatty liver and hepatoma.