重组腺相关病毒Ⅰ型/大鼠肿瘤坏死因子受体:Fc(rAAVⅠ/ratTNFR:Fc)的构建和在大鼠体内表达的初步研究

Construction and in vivo Expression of Recombinant Adeno-associated Virus Type-Ⅰ in Rats Vector Encoding Rat Tumor Necrosis Factor Receptor: Fc Fusion Gene (rAAVⅠ/ratTNFR:Fc

目的 构建携带大鼠肿瘤坏死因子Ⅱ型受体胞外区和大鼠免疫球蛋白IgG1Fc段融合基因的重组Ⅰ型腺相关病毒(rAAVⅠ/ratTNFR :Fc) ,并对该病毒在Lewis大鼠体内的表达和生物学活性进行初步研究,探讨以该重组腺相关病毒载体应用于类风湿关节炎基因治疗的可行性。方法 首先以RT- PCR分别从大鼠脾细胞和大鼠淋巴细胞总RNA中扩增出大鼠肿瘤坏死因子Ⅱ型受体胞外区和大鼠免疫球蛋白IgG1Fc段基因,并以重叠延伸PCR的方法将二者融合后克隆入pSNAVⅠ载体质粒进行重组病毒生产。在获得重组病毒后于Lewis大鼠肌肉注射,于不同时间ELISA测定血清ratTNFR :Fc表达,并以TNF -α细胞毒中和实验测定重组病毒表达产物的生物学活性。结果 所构建的重组病毒rAAVⅠ/ratTNFR :Fc基因结构与预期一致;病毒于Lewis大鼠肌肉注射后可表达ratTNFR :Fc蛋白,含有ratTNFR :Fc蛋白的血清可以有效中和大鼠TNF- α对L92 9细胞的杀伤作用。结论 本研究所构建的携带大鼠肿瘤坏死因子Ⅱ型受体胞外区和大鼠免疫球蛋白IgG1Fc段融合基因的重组Ⅰ型腺相关病毒(rAAVⅠ/ratTNFR :Fc)可以用来作为阻断TNF α的手段于大鼠类风湿关节炎模型上进行基因治疗研究。

Objective To investigate the feasibility of rheumatoid arthritis gene therapy mediated by adeno-associated viral vector, a recombinant adeno-associated virus type-Ⅰ vector encoding rat tumor necrosis factor receptorⅡextracellular region and rat IgG_1 Fc fragment fusion gene (rAAVⅠ/ratTNFR:Fc) was constructed. Biological activity of ratTNFR:Fc and virus expression in rat were investigated. Methods Rat tumor necrosis factor receptorⅡextracellular region gene and Fc gene fragment of rat IgG_1 were amplified by RT-PCR from total RNA of rat spleen cell and rat lymphocytes respectively. Then the ratTNFR:Fc fusion gene obtained by overlapping PCR was cloned to general vector pSNAVⅠ for rAAV production. Rats received rAAVⅠ/ratTNFR:Fc by muscle injection after virus production.Biological activity of rAAVⅠ/ratTNFR:Fc and fusion gene expression in vivo were identified by TNF-α neutralization assay and ELISA respectively at various. Results Genetic structure of rAAVⅠ/ratTNFR:Fc was confirmed according to the plan. ratTNFR:Fc protein was identified in rat serum, and this ratTNFR:Fc protein containing serum could effectively suppress the cytotoxicity in L929 cell induced by rat TNF-α in vitro. Conclusion This study shows that the rAAVⅠ/ratTNFR:Fc constructed by us, can be used as anti-inflammation gene therapy for rheumatoid arthritis through TNF-α pathway blockage.