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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。
Objective: To establish a model of H9 cells (human CD4, T cells) transfected with recombinant HCV gene. Methods: The high fidelity 1.73kb DNA fragment (including 1693bp HCV cDNA and partial vector DNA sequence) was obtained by PCR using the vector CDZ2 (with 1693 bp of HCV DNA, including the complete HCV structural region) as a template whose specificity was confirmed by southern blot. The HCV cDNA fragment was inserted into the vector pCD-SRα, and the recombinant HCV (pCD-HCV) was obtained by colony in situ hybridization and digestion with southern blot. The recombinant plasmid was transfected into H9 cells with pSV2-gpt vector. RESULTS: Oral positive clonal cells were screened from selective media by RT-PCR. DotELISA and western blot showed that pCD-HCV could be replicated, transcribed and expressed in H9 cells. The expressed protein of about 1.3 × 105 was HCV antigenic. HCV mRNA and HCV antigens could still be detected when cells transfected with pCD-HCV were cultured to 90 days. Conclusion: The establishment of pCD-HCV transfected H9 cell model was successful.