Expression Of Hepatitis C Virus H Strain Envelope Glycoprotein In Insect Cells And Detection Of Anti-E Antibodies In HCV Infected Serum

Expression of hepatitis C virus H strain envelope glycoprotein in insect cells and detection of anti-E antibodies in HCV infected serumM.D. studentFu liDirectorProfessor XU Zhi-kaiAssistant InstructorProfessor XUE Xiao-pingHepatitis C virus (HCV) is a major causative agent of post-transfusion and community-acquired hepatitis in the world. The majority of HCV-infected individuals develop chronic hepatitis progressing eventually to liver cirrhosis and hepatocellular carcinoma, neither an effective treatment for chronic HCV infection nor a vaccine to prevent HCV infection is available at the present time. The only available treatment, a combination of alpha interferon and ribavirin, is efficacious in only a minority of patients. The development of such therapies will be aided greatly by a more complete picture of the structure-function features of HCV proteins.HCV is an enveloped plus-strand RNA virus of the Flaviviridae family. Its genome is 9.4 kb in length with one open reading frame that is translated as a single polyprotein, which is processed by host and virus proteases into at least three structural and seven non-structual proteins with various enzymatic activities. Two glycoproteins are probably virion envelope proteins, which are obvious candidates for inclusion in a subunit vaccine because of its potential rolein HCV attachment.In this research, we constructed the transfer plasmid E-BacPAK8 which contains the 5'-terminal 1575 bp segments of HCV E gene in H strain. Then, we transfered it with digested BacPAK6 viral DNA into insect cells Sf9 and expressed the glycoprotein. According to the SDS-PAGE results, the molecular weight of expressed envelope protein(gE) was similar with other reports. We observed that there're several protein bands on Coomassie Blue gel staining, which include 60 000, 35000, 33 000^ 26 000 and 22 000. Using serum from HCV RNA positive patient ,we studied the pattern of HCV-specific immunoreactive proteins produced by the recombinant virus. On Western blot we detected a 60000 protein, which was EI , and a series of closely spaced bands between 35000 and 22000 representing EI . A 31000 band is a nonspecific protein which was displayed both in normal Sf9 and Sf9 infected by BacPAK6. Finally, we detected the anti-E antibodies in 35 sera from HCV-infected patients using expressed gE. The detected ratio of anti-E antibody in HCV positive sera was very low, only 4 of 35 patients were anti-E antibody positive. In addition, the ratio of anti-E antibody in HCV core protein antibody positive sera was lower than that of RNA positive ones.