TARGET CELL SPECIFIC GENE TRANSFER BY ANTIFECTION AND REGULATION OF PROLIFERATION OF CANCER CELLS

目的：为了开发一种抗体介导的靶向基因转染新技术--抗感染方法，我们确定了基因转移的最佳条件。材料与方法：用苯醌将抗CD 3抗体OKT 3化学偶联到含有绿色荧光蛋白（GFP）基因的质粒DNA上。通过将混合物在40%蔗糖溶液上分层，然后进行超离心来富集Ab-DNA缀合物。DNA和Ab之间的偶联通过1%琼脂糖凝胶电泳，然后通过抗小鼠IgG抗体进行蛋白质印迹来确定。结果与讨论：琼脂糖凝胶电泳显示，与单独的质粒DNA相比，Ab-DNA偶联物的电泳迁移率发生了变化。Western印迹显示抗小鼠IgG抗体与Ab-DNA反应混合物中观察到的条带反应，表明Ab-DNA缀合物的存在。通过将脂质（FuGENE）偶联到Ab-DNA缀合物上，观察到Ab-DNA缀合物成功转染到Jurkat细胞中。氯喹由于其细胞毒性而未能赋予成功的转染。抗转染可能是基因治疗的一种有效方法。

Purpose : To develop an antifection method, a new technique of antibody-mediated targeted gene transfection, we determined optimal conditions of the gene transfer.Materials and Methods : OKT3, the antibody (Ab) against CD3 was chemically conjugated using benzoquinone to pEGFP, plasmid DNA containing green fluorescent protein (GFP) gene. The Ab-DNA conjugate was enriched by layering the mixture on a 40% sucrose solution followed by an ultracentrifugation. Coupling between DNA and Ab was determined by a 1% agarose gel electrophoresis followed by western blotting by anti-mouse IgG antibody. Jurkat T cells expressing CD3 were incubated with the Ab-DNA conjugate, and then determined the expression of GFP by flowcytometry.Results and Discussion : Agarose gel electrophoresis showed a change of the electrophoretic mobility in the Ab-DNA conjugate, as compared to plasmid DNA alone. The western blot revealed that anti-mouse IgG antibody reacted against the observed band in the Ab-DNA reaction mixture, indicating the existence of the Ab-DNA conjugate. Successful transfection of the Ab-DNA conjugate into Jurkat cells was observed by coupling lipids (FuGENE) to the Ab-DNA conjugate. Chloroquine failed to confer successful transfection due to its cytotoxicity. Antifection could be an useful method for gene therapy.