Full length NS5A gene of the hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural gene as template. The amplified fragment (about 1.34 kb) was cloned into plasmid pQE32, and the recombinant plasmid pQENS5A was expressed in JM109 strain. The NS5A protein was purified by NiSO 4 metal chelating resin, and characterized by Western blot. Its antigenecity was determined by ELISA. The positive detection rate of anti NS5A was 75% (69/92) in ninety two clinic sera. The positive rate of anti NS5A was 82.5% (33/40) in fourty positive standand sera, and the negative rate of anti NS5A was 100% (40/40) in fourty negative standand sera. The results showed that the Full length NS5A proteinn had the higher sensitivity and specificity in the detection of HCV antibody in sera, we suggested that NS5A protein was a useful antigen for blood screening.
Full length NS3 gene of a hepatitis C virus was amplified by PCR using plasmid pBAC25 containing HCV nonstructural protein gene as template. The amplified fragment (about 1.8 kb) was cloned into plasmid pQE30 and the recombinant plasmid was expressed in JM109. The NS3 protein was purified by NiSO 4 metal chelating resin, and its antigenecity was determined by ELISA, the results showed that the full length NS3 protein was more sensitive than the commercial carboxy terminal domain of NS3 protein in HCV antibody detection.
采用 PCR技术获得了新型的人肿瘤坏死因子基因 ,在 15 3位氨基酸处引入突变 ,使 Gly突变为 L eu.将突变体 h TNF基因克隆到表达载体 p QE30中 ,SDS- PAGE结果显示经 IPTG诱导后 ,h TNF基因获得大量表达 .通过 Ni金属鳌合层析柱亲和层析获得到纯化的 h TNF融合蛋白 ,Western blot结果表明 h TNF蛋白可与抗TNF的抗血清发生特异性反应 .荧光染色实验检测了 h
