重组丙型肝炎病毒基因转染H9细胞模型的建立

ESTABLISHMENT OF H9 CELL MODEL TRANSFECTED BY RECOMBINANT HEPATITlS C VIRUS

目的：建立重组ＨＣＶ基因转染的Ｈ９细胞（人ＣＤ４，Ｔ细胞）模型。方法：以载体ＣＤＺ２（连接有１６９３ｂｐＨＣＶＤＮＡ，包括完整的ＨＣＶ结构区）为模板，通过ＰＣＲ获得高保真的１．７３ｋｂＤＮＡ片段（包括１６９３ｂｐＨＣＶｃＤＮＡ和部分载体ＤＮＡ序列），其特异性被ｓｏｕｔｈｅｒｎｂｌｏｔ证实。将ＨＣＶｃＤＮＡ片段插入载体ｐＣＤ－ＳＲα，经菌落原位杂交以及酶切分斤和ｓｏｕｔｈｅｒｎｂｏＩｔ筛选，获得重组ＨＣＶ（ｐＣＤ－ＨＣＶ），并与ｐＳＶ２－ｇｐｔ载体共转染Ｈ９细胞。结果：从选择性培养基中筛选口阳性克隆细胞，经ＲＴ－ＰＣＲ．ｄｏｔＥＬＩＳＡ和ｗｅｓｔｅｒｎｂｌｏｔ验证表明ｐＣＤ－ＨＣＶ在Ｈ９细胞中可进行复制、转录和表达，表达的约１．３×１０５大小蛋白具有ＨＣＶ抗原性。转染ｐＣＤ－ＨＣＶ的细胞培养至９０天时，仍可检测到ＨＣＶｍＲＮＡ和ＨＣＶ抗原。结论：建立ｐＣＤ－ＨＣＶ转染的Ｈ９细胞模型获得成功。

Aim: To establish H9 cell (a human CD+ T cell line) model transfectecl by recombinant HCV. Methods: 1 693bp HCV cDNA (total structure vegion gectes) was acquired by PCR, the specificity was verified by southern blot. The HCV cDNA was inserted to pCD-SRα vector by ligation reaction. To identify the recombinant HCV, it was transformed into JM109 E.coli strain, then in situ hybridization, restriction enzyme reaction and southem blot were carried out respectively. The recombinant HCV of correct sequence was named pCD-HCV. which was cotractsfected into H9 cell line with pSV2-gpt DNA vector. The cells were cultured in XHTA-M selective culture medium. RT- PCR, dot ELISA and western blot were manipulated. Results: The construction of pCD-HCV was successful. pCD-HCV was replicated, transcribed and expressed in the H9 cells. The molecular weight of the expressed HCV protein is about130kDa. After cultured for 90 days, the results of RT-PCR and dot ELISA showed that pCD-HCV still transcribed and exlpressed. Conclusion : The H9 cell model transfected by pCD-HCV was maintaiated three months in the culture medium .