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 rec...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.展开更多
应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p Q...应用 PCR方法从含有丙肝病毒全长非结构蛋白基因的载体 p Blue Bac2 5中扩增出全长的 N S2基因DNA片段 ,分别克隆到表达载体 p QE3 0和转座载体 p Fast Bac HTb的多克隆位点 ( MCS) .PFast NS2通过转座插入穿梭载体 Bacmid的表达盒 ;p QENS2转化 JM10 9菌株 ,诱导表达出 N端含有 6个 His的全长 NS2蛋白 ,用 Ni-NTA- agarose柱层析纯化 ,获得提纯的全长 NS2蛋白 .展开更多
文摘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.
文摘采用 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
