丙型肝炎病毒不同编码区His融合抗原的表达与纯化

目的:表达和纯化丙型肝炎病毒(HCV)-Core、NS3和NS4的His融合抗原H-C、H-NS3和H-NS4,并初步探讨其在抗体检测中的应用。方法:用重组基因工程技术,构建3种重组质粒C-pET-28a-c( )、NS3-pET-28a-c( )和NS4-pET-28a-c( )以及相应的工程菌;质粒经双酶切、PCR和测序鉴定正确后,IPTG诱导抗原表达,从IPTG使用浓度、诱导温度与时间3个方面优化抗原表达条件;用NTA柱纯化抗原后,分别用单片段重组抗原和混合抗原以酶联免疫吸附试验(ELISA)检测100份HCV抗体阳性血清和40份健康人血清。结果:用单片段重组抗原和混合抗原检测的100份HCV抗体阳性血清中,H-C、H-NS3和H-NS4的抗体阳性率分别为91%、75%和52%,而H-C、H-NS3和H-NS4混合抗原的抗体检出率为100%;检测40份健康人血清,结果全部为阴性。结论:HCV不同编码区单片段His融合抗原具有不同的抗体检出率,但均低于混合抗原的阳性率;在开发HCV抗体诊断试剂时,应采用HCV混合抗原。

Expression and purification of His-tag recombinant antigens encoded by a series of fragments of HCV

Objective To express the His-tag recombinant proteins encoded by a series of fragments of hepatic C virus (HCV) gene and to explore the significance of the proteins in the detection for anti-HCV antibodies.Methods Three recombinant plasmids C-pET-28a-c ( ),NS3-pET-28a-c ( ) and NS4-pET-28a-c( )] were constructed using the techniques of gene engineering.The recombinant plasmids were confirmed by PCR,endonuclease splicing and sequencing.Three His-tag recombinant antigens (H-C,H-NS3 and H-NS4) were expressed by the induction of IPTG and purified with Ni2 -NTA resin,which were applied for ELISA detection of anti-HCV antibodies.100 serum samples which were known to be anti-HCV-positive were tested with each of the three antigens and the mixed proteins.40 specimens collected from healthy persons were also tested with the antigens.Results The positive rates of anti-C,anti-NS3,and anti-NS4 in the anti-HCV-positive sera were 91%,75%,and 52%,respectively.However,all the anti-HCV-positive sera were immunoreactive to the mixed antigens,and all the specimens obtained from healthy persons did not react to the antigens.Conclusions There are different immunoreactivities of the His-tag recombinant antigens encoded by different gene fragments.The mixture of all the antigens derived from HCV different encoding regions should be applied in further HCV diagnostic test to detect anti-HCV antibodies.