一种新型抗体导向核酸转移系统关键组分的制备及鉴定  

Preparation of SPA-PLL conjugate as a key component for construction of a novel antibody-targeted non-virus gene delivery system

作　　者：刘晓芬[1] 刘云[2] 张萃[1] 吴强[1] 肖笑荣 刘晓波[1] 

机构地区：[1]广东药学院基础学院,广东广州510006 [2]川北医学院基础医学院,四川南充637000

出　　处：《广东药学院学报》2015年第3期372-377,共6页Academic Journal of Guangdong College of Pharmacy

基　　金：教育部留学回国人员启动基金(44143006);四川省教育厅自然科学重点项目(13ZA0220);广东省自然科学基金项目(010763)

摘　　要：目的制备并鉴定葡萄球菌A蛋白-多聚赖氨酸交联物(SPA-PLL交联物),以此交联物为"接头",构建一种新型的抗体导向核酸转移系统。方法采用SPDP化学偶联法制备SPA-PLL交联物;采用SDSPAGE电泳法测定SPA-PLL交联物的纯度;采用酶联法或DNA凝胶阻滞实验分别测定SPA-PLL交联物与IgG或DNA片段的结合;将SPA-PLL交联物与Tfr抗体和p EGFP-C1质粒以适宜的质量比混合即可组装成Tfr抗体导向pEGFP-C1质粒转移系统;将该抗体导向的pEGFP-C1质粒转染人肝癌HepG2细胞,在荧光显微镜下观察绿色荧光蛋白(GFP)的表达。结果 SPA-PLL交联物纯度较高,能良好地结合多种Ig G抗体,同时也能较好地结合pEGFP-C1质粒、中和其负电荷;Tfr抗体导向pEGFP-C1质粒转移系统能特异性地转染Hep G2细胞,并使pEGFP-C1质粒在细胞中有效表达。结论 SPA-PLL交联物能与核酸片段和IgG抗体非共价结合,并具有良好通用性;以此制备抗体导向核酸转移系统的过程简单方便。Objective To prepare SPA-PLL conjugate as a component or adaptor for construction of a novel antibody-targeted gene delivery system. Methods SPA-PLL conjugate was prepared by SPDP chemically crosslinking technique and its purity was measured by SDS-PAGE electrophorisis. The bindings of SPAPLL conjugate to Ig G and DNA were assayed by ELISA and gel retardation assay,respectively. Tfr-specific antibody-targeted p EGFP-C1 plasmid delivery system was constructed by mixing SPA-PLL conjugate with p EGFP-C1 plasmid and Tfr-specifc antibody at room temperature for 60 min. Expression of the plasmid in Hep G2 cells was analyzed by fluorescence microscope. Results The SPA-PLL conjugate had a higher purity and bound to many kinds of Ig Gs and p EGFP-C1 plasmid. Tfr-specific antibody-targeted p EGFP-C1 plasmid delivery system was specifically transfected and expressed in Hep G2 cells. Conclusion SPA-PLL conjugate could non-covalently bridge Ig G antibody and DNA for construction of the antibody-targeted gene delivery system.

关 键 词：葡萄球菌A蛋白(SPA) 多聚赖氨酸(PLL) IgG 核酸 基因转染 

分 类 号：R392[医药卫生—免疫学]