Tumor cell-specific gene transfer with retroviral vectors displaying single-chain antibody  

单链抗体-包膜蛋白嵌合型单嗜性逆转录病毒构建及靶向感染肿瘤细胞的研究(英文)

作　　者：汤宇澄[1] 李煜[1] 钱关祥[1] 

机构地区：[1]Department of Biochemistry and Molecular Biology,the Researeh Center for Human Gene Therapy,Shanghai Second Medical University,Shanghai 200025,China

出　　处：《Chinese Medical Journal》2002年第7期1064-1069,153,共6页中华医学杂志（英文版）

基　　金：ThisresearchissupportedbyNationalScienceFoundationofChina(No .863 Z2 0 0 1 0 4)

摘　　要：OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing plasmid.To confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. RESULTS: The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. CONCLUSION: These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.目的　将单链抗体基因插入单嗜性逆转录病毒胞膜蛋白 ,以构建能靶向感染肿瘤细胞的逆转录病毒载体。方法　构建单链抗体融合表达质粒PET2 0b scFv ,通过酶联免疫分析 ,Westernblot检测基因工程单链抗体的抗原结合活性。利用PCR定点突变技术 ,在野生型MoMulv胞膜蛋白上的SU亚单位氨基末端产生合适的酶切位点 ,插入体外构建 ,证实具有抗原亲合活性的抗人脑胶质瘤细胞膜抗原的单链抗体基因 ,并构建利用CMV启动子高效表达胞膜蛋白 单链抗体融合基因的表达质粒 ,转染Ψ2包装细胞后 ,以lac z为报告基因 ,包装出重组逆转录病毒 ,进行病毒结合实验和感染实验。结果　酶联免疫分析 ,Westernblot结果证实基因工程单链抗体能与肿瘤细胞表面相应抗原结合 ,病毒结合实验与感染实验证明所构建嵌合型逆转录病毒能改变其嗜性 ,由仅感染鼠细胞到特异性感染人脑胶质瘤细胞 ,且这种作用能为特异性单克隆抗体阻断。结论　利用单链抗体修饰单嗜性逆转录病毒胞膜蛋白的SU亚单位能达到选择性感染肿瘤细胞的目的。

关 键 词：Gene Therapy Gene Transfer Techniques Genetic Vectors Hela Cells Humans Immunoglobulin Fragments NEOPLASMS Research Support  Non-U.S. Gov't RETROVIRIDAE 

分 类 号：R346[医药卫生—基础医学]