重组腺相关病毒2型/人肿瘤坏死因子受体:Fc(rAAV2/hTNFR:Fc)的构建和生物学活性研究

为研究腺相关病毒2型载体应用于类风湿性关节炎进行基因治疗的可行性，首先构建携带人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段融合基因的重组2型腺相关病毒(rAAV2／hTNFR：Fc)，并对其生物学活性进行研究。以RT—PCR分别从U937和人淋巴细胞总RNA中扩增人肿瘤坏死因子Ⅱ型受体胞外区和人免疫球蛋白IgG1Fc段基因，并以重叠延伸PCR的方法将二者融合后克隆入pSNAV1载体质粒进行重组病毒生产，在进行重组病毒理化分析后，以TNFa细胞毒中和试验来研究该重组病毒的生物学活性。结果显示：所构建的重组病毒rAAV2／hTNFR：Fc基因结构与预期一致；病毒在体外感染BHK-21细胞后，含TNFR：Fc融合蛋白的表达上清可以有效中和人、大鼠、小鼠TNFα对L929的细胞毒性。所研究构建的重组腺相关病毒可以用来作为阻断TNFα的手段，进行类风湿性关节炎的基因治疗研究。

Construction and Biological Activity Research of Recombinant Adeno-associated Virus Type-2 Vector Encoding Human Tumor Necrosis Factor Receptor: Fc Fusion Gene (rAAV2/hTNFR: Fc)

To investigate the feasibility of rheumatoid arthritis gene therapy mediated by adeno-associated viral vector, a recombinant adeno-associated virus type-2 vector encoding human tumor necrosis factor receptor extracellular region and human IgG1 Fc fragment fusion gene (rAAV2/hTNFR:Fc) was constructed. Human tumor necrosis factor receptorextracellular region gene and Fc gene fragment of human IgG1 were amplified by RT-PCR from total RNA of U937 cell and human lymphocytes respectively. Then the hTNFR:Fc fusion gene obtained by overlapping PCR was cloned to general vector pSNAV1 for rAAV production. We identified the biological activity of rAAV2/hTNFR:Fc by TNF neutralization assay in vitro after virus purification and physical characteristics analysis. Genetic structure of rAAV2/hTNFR:Fc was confirmed as the plan. The hTNFR:Fc protein contained supernatant of BHK-21cell infected by rAAV2/hTNFR:Fc could effectively suppress the cytotoxicity in L929 cell induced by TNF of human, rat or mouse origin in vitro. This study showed that the rAAV2/hTNFR:Fc,we constructed, can be used as anti-inflammation gene therapy for rheumatoid arthritis through TNF pathway blockage.