丙肝病毒非结构4区和5区抗原的克隆表达及纯化鉴定

Expression of HCV NS_4 and NS_5 Gene Fragments and Identification of the Expressed Proteins

通过分析丙肝病毒（ＨＣＶ） 的非结构４ 区（ＮＳ４） 和５ 区（ＮＳ５）蛋白的氨基酸序列，推测确定抗原决定簇位点。用ＲＴ－ ＰＣＲ 技术从中国丙肝病人血清中扩增克隆含抗原决定簇基因的ＮＳ４ 及ＮＳ５ 基因，将该两段基因分别克隆至质粒表达载体ｐＥＴ２８ａ（ ＋ ） 内，转化大肠杆菌ＢＬ２１（ＤＥ３） ，构建成功了高效表达ＮＳ４ 和ＮＳ５ 蛋白的工程菌，ＩＰＴＧ 诱导后两蛋白在工程菌内的表达量分别约占菌体蛋白的３３ ％ 和２８ ％ 。经Ｓｅｐｈａｒｃｒｙｌ Ｓ－ ２００ 分子筛及Ｎｉ－ ＮＴＡＳｅｐｈａｒｏｓｅ 螯合层析纯化，获得了较纯的重组ＮＳ４ 和ＮＳ５ 蛋白。用纯化的ＮＳ４ 和ＮＳ５ 蛋白作抗原ＥＬＩＳＡ 法检测ＨＣＶ抗体阴、阳性血清，证实它们有较好的抗原性和特异性。

The NS 4 and NS 5 gene fragments of hepatitis C virus were cloned from anti HCV positive sera by RT PCR, the recombinant plasmids expressing NS 4 or NS 5protein were constructed by inserting the cloned NS 4 or NS 5 gene fragments into plasmid vector pET28a(+), the expressed NS 4 protein is about 33% of total host ( E.coli BL21) proteins, and the NS 5 is about 28%. After purification, the expressed NS 4 and NS 5 protein were used to detect 87 anti HCV (anti HCV core and or NS 3 antigen) positive sera and 160 anti HCV(anti core and NS 3 antigens) negative sera, of the 87 positive sera, 28 (31%) sera are anti NS 4 positive and 62(70%) sera are anti NS 5positive; of the 160 negative sera, 2 sera are anti NS 4 weak positive and 1 serum is anti NS 5 weak positive. The results suggest that the NS 4 and NS 5 proteins have excellent antigenicity and specificity, and may increase the detection rate of anti HCV when being added in ELISA kit for detection of anti HCV.