Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified a...Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.展开更多
The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid ...The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid were expressed in E.coli 06. The pHC CN plasmid expressed fussion protein was shown by a major band with a expected molecular weight about 68 kD on SDS PAGE. However, the pHCV NC plasmid expressed fussion protein was 66 kD in molecular weight. The results indicate that the pHCV NC fussion protein differs from the pHCV CN fussion protein in mulecular weight. It suggests that the fusion way is important for the structure of recombinant proteins.展开更多
文摘Objective To express the hepatitis C virus (HCV) core gene in E . coli on a high level. Methods The cDNA coding for HCV core protein was amplified by polymerase chain reaction (PCR). The PCR product was purified and digested with restriction enzymes and inserted into the downstream of P RP L promoter of a high level expression vector pBV220 . HCV core gene was expressed in E . coli in a non fused form. The expression protein was analysed by SDS PAGE , and its immunoactivity was tested by ELISA . Results Sequence analysis of the amplified PCR products confirmed that we have successfully cloned and expresssed the intact core protein of HCV. SDS PAGE showed that a specific protein with a molecular weight of 21kDa at a level of 14.0% of the total bacterial proteins appeared in bacteria harboring pBV/HCVCore, while this protein was absent in the control bacteria harboring pBV220. The results of enzyme immunoassay analysis showed that this protein could be specifically recognized by the HCV positive sera from patients with hepatitis C .Conclusion The intact HCV core protein was successfully expressed in E . coli in a non fused form on a high level, and its immunoactivity was high.
文摘The gene encoding the core and NS3 proteins of hepatitis C virus was amplified by PCR, respectirely. Two genes were fused and formed the recombinant plamid pHCV CN (Core+NS3) and pHCV NC (NS3+Core). The fused plasmid were expressed in E.coli 06. The pHC CN plasmid expressed fussion protein was shown by a major band with a expected molecular weight about 68 kD on SDS PAGE. However, the pHCV NC plasmid expressed fussion protein was 66 kD in molecular weight. The results indicate that the pHCV NC fussion protein differs from the pHCV CN fussion protein in mulecular weight. It suggests that the fusion way is important for the structure of recombinant proteins.
