丙型肝炎病毒基因组C区和NS3区基因cDNA的融合及其在大肠杆菌中的表达与初步应用

cDNA CHIMERIC FUSION AND PROKARYOTIC EXPRESSIONOF NUCLEOCAPSID AND NS3 REGIONS OF HEPATITIS CVIRUS GENOME AND ANALYSIS ITS ANTIGENICITY

通过逆转录（ＲＴ）－聚合酶链式反应（ＰＣＲ），从中国人丙型肝炎病毒（ＨＣＶ）携带者的血清中扩增并克隆到２段ｃＤＮＡ片段，即ＨＣＶ基因组Ｃ区抗原基因Ｃ８３１ｃＤＮＡ片断（约５３０ｂｐ）和ＮＳ３区抗原基因Ｃ３３ｃｃＤＮＡ片段（约８６０ｂｐ）。Ｃ３３ｃｃＤＮＡ片段同Ｃ８３１ｃＤＮＡ片段经连接 肽Ｓｅｒ－Ｐｒｏ－Ｇｌｙ－Ｓｅｒ连接成为基因嵌合体Ｃ３３ｃ－Ｃ８３１（约１４００ｂｐ）。Ｃ３３ｃ－Ｃ８３１基因嵌合体同温控型原核表达载体ｐＢＶ２２０重组，构建成表达质粒ｐＢＶ／Ｃ３３ｃ－Ｃ８３１，并在大肠杆菌细胞中获得了重组嵌合抗原Ｃ３３ｃ－ＣＬ的表达。通过酶切分析和Ｗｅｓｔｅｒｎ免疫印迹法，对约占菌体可溶性蛋白９％的表达产物做了鉴定。采用ＴｒｉｔｏｎＸ－１００和盐析处理，获得粗提表达产物。粗提的表达产物经尿素裂解和离子交换层析纯化，得到可用于检测抗ＨＣＶ核壳蛋白和抗ＮＳ３区抗体的重组嵌合抗原Ｃ３３ｃ－ＣＬ。对Ｃ３３ｃ－ＣＬ做抗原性分析发现，它同时具有完整的Ｃ３３ｃ抗原和Ｃ２２抗原的免疫反应活性，完全能替代单纯的Ｃ３３ｃ和Ｃ２２抗原。该嵌合抗原在血清学诊断中有重要的应用价值，可望成为新一代ＨＣＶＥＩＡ诊断试剂的优选抗原。

DNA fragments of C831(530bp)and C33c (860bp)encoding the putative nucleocapsid(C)CL and the nonstructural region 3(NS3) protein C33c of the HCV, have been obtained fromthe sera of Chinese carriers with HCV infection by reverse transcription (RT)and polymerasechain reaction(PCR) techniques. The 5' terminal of C831 cDNA fragment was linked upup with the 3'terminal of NS3 cDNA fragment by a oligonucleotide linkerSer-Pro-Gly-Ser to form a chimeric gene C33c-C831(1400bp)The chimeric geneC33c-C831 was recombined with prokaryotic expression vector pBV220,and was expressed inE. coli in the form of a native chimeric polyprotein C33c-CL.The expression product wasscreened and detected by enzyme-linked immune solid assay and western blotting withanti-C33c serum and anti-CL serum,respectively.A chimeric C33c-CL polyprotein with amolecular weight of 53kD was acounted for 9% of the total cellular soluble proteins.The expression product was extracted from the bacterial lysate by lysozyme, Triton X-100and urea treatment and purified by ion exchange chromatography. The purified C33c-CLchimeric polyprotein was used to develop a capture assay for reactive anti-HCV antibodies.This anti-C33c-CL assay detected all previously identified HCV-seropositive cases andprovides a substantially more sensitive diagnosis(99%)than any of anti-C33c(93%)anti-C22(83%)or anti-5-1-1(50%) for both acute and chronic HCV infections. TheC33c-CL chimeric polyprotein keeps the antigenicity of both C33c and CL. Its specificityand sensitivity were all in keep with the requirements of the national standard for qualitycontrol of the HCV diagnostic kit.The C33c-CL chimeric polyprotein may have animportant role in anti-HCV assay.