【摘要】 本文通过逆转录（ＲＴ）－聚合酶链式反应（ＰＣＲ），从两份分别来自湖南省娄底地区丙型肝炎病人和河北省秦皇岛市职业献血员丙型肝炎病毒（ＨＣＶ）ＲＮＡ打点杂交阳性的血清中，扩增并克隆到１段５６３ｂｐ的ＨＣＶ基因组Ｃ区抗原基因Ｃ２６９／８３１，并通过ＰＣＲ得到了３个表达片段Ｃ８３１、Ｃ８０１和Ｃ５８７。测定Ｃ２６９／８３１基因的全序列后发现，中国人ＨＣＶ湖南分离株与ＨＣＶ－Ⅰ型株ＨＣＶ－ＵＳ和Ⅱ型株ＨＣＶ－ＢＫ在该基因区段的核苷酸／氨基酸序列的同源性，分别为９０．３％／９４．６％和９５．２％／９４．６％。利用原核高效表达载体ｐＢＶ２２０在大肠杆菌中有效地表达了非融合的Ｃ区抗原基因重组蛋白ＣＬ、ＣＭ和ＣＳ。通过免疫筛选法及Ｗｅｓｔｅｍ印迹法对约占菌体可溶性蛋白１１％的表达产物进行了鉴定。采用ＴｒｉｔｏｎＸ－１００和盐析处理表达产物，再进行离子交换层析纯化，得到可用于检测ＨＣＶ血清抗体的核壳蛋白（Ｃ）抗原。通过不同分子量抗原的表达，发现由Ｃ区Ｎ端８９个氨基酸组成的多肽ＣＳ其抗原性与由１５８或１６８个氨基酸组成的多肽ＣＭ或ＣＬ相同，但抗原的稳定性和表达量显著优于后两种抗原。本研究为研制ＨＣＶ抗体诊断试剂盒奠定了重要基础。更多还原

【Abstract】 cDNA fragment of C269/831(560bp)and three cDNA fragments of C831,C801,C587corresponding to the putative nucleocapsid (C)of the HCV genome were obtained from the seraderived from Chinese carriers with HCV infection,who came from Loudi of Hunan provinceand Qinghuangdao of Hebei province of China , by application of reverse transcription (RT)and polymerase chain reaction (PCR)technique.As the sequence of the cDNA fragment ofC269/831(HCV-Hunan Strain)was determined and compared with those of the corresponding regions of the HCV-Ⅰ(HCV-US )and HCV-Ⅱ(HCV-BK)genomes,the nucleotide/amino acid sequence homologies to HCV-US and HCV-BK isolates were found to be 90.3%/94.6%and 95.2%/94.6%,respectively.The prokaryotic expression vector pBV220 was employed for the overproduction of CL,CM and CS native recombinant proteins in E.coli cellderived from C831, C801 and C587 cDNA fragments.The expression products were screenedby bacterial clony hybridization ,antibody -sandwich enzyme immunoassay,and determined by Western blotting with antisera of chronic hepatitis C patients. The 20KD recombinant pro-teins CL, CM and the 14KD of recombinant protein CS were shown to account for about 11%of the total cellular soluble proteins。The recombinant expression products were ex-tracted from the bacterial lysates by lysozyme ,Triton X-100and urea treatment ,and purified through the ion exchange chromatography .The purified recombinant nucleocapsid protein CSwas used to capture and measure reactive antibodies both acute and chronic HCV infec-tions.THe study indicates that at the nucleotide level ,the C269/831 cDNA sequence homology(95.2%)between the HCV -Hunan isolate and the HCV-BK isolate is higher than the homology (90.3%)between the HCV -Hunan isolate and HCV-US isolate,but at the aminoacid level ,the sequence homogy (94.6%)is similar between the HCV-Hunan isolate and both HCV-BK or HCV-US isolate In addition,the HCV- Hunan is significantly diverse fromother HCV isolates ,which proves once more that the HCV genome is heterogeneous among the differet isolates derived from different areas.More conservative is the amino acid sequence of the N terminal two hydrophilicities of C region of the HCV genome among the all different HCV isolates ,which suggests that the N terminal two hydrophilicities of C region of the HCVgenome would have an important biological function,and would be an appropriate antigen for the HCV serodiagnosis.There is 99%of over lapping immunoreactitivity between the total nucleocapsid region and the CS,which is composed of 89 amino acods pf tje N terminaltwo hydrophilicities of C region of the HCV genome .As for the expression level and the antigen stability,recombinant protein CS was found better than CL and CM.更多还原