丙型肝炎病毒不同编码区His融合抗原的表达与纯化

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

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。

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 Ni^2＋ -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 HCV fragments. The mixture of all the antigens derived from HCV different encoding regions should be applied in further diagnostic test to detect anti-HCV antibodies.