一种新型的具有免疫调节和介导DNA基因治疗的蛋白质载体的研制

Development of a Novel Protein Carrier Inducing Immune Response and Binding DNA in Gene Therapy

目的:研制一种具有调节宿主免疫系统,同时又能包装核酸进入肿瘤细胞的新型蛋白质载体。方法:利用基因工程技术,将人乙肝病毒核心抗原(HBcAg)的-COOH端和靶向肿瘤的整和素配体RGD肽与谷胱甘肽还原酶(GST)在大肠埃希菌中融合表达,经分子筛和GST亲和纯化后,获得DNA包装蛋白GST-RGD-ΔHBcAg。此包装蛋白用FITC标记后与前列腺癌PC-3细胞孵育,观察进入细胞情况;并用此包装蛋白包装含绿色荧光蛋白GFP基因的DNA载体pEGFP-N1,转染PC-3细胞,观察绿色荧光蛋白表达。此外,用包装蛋白GST-RGD-ΔHBcAg免疫6周龄雌性BALB/c小鼠,检测血清IgG1和IgG2a抗体水平。结果:本研究应用原核表达系统成功地表达并纯化出DNA包装蛋白GST-RGD-ΔHBcAg;荧光显微镜下观察到包装蛋白GST-RGD-ΔHBcAg能进入到PC-3细胞,并能携带绿色荧光蛋白基因进入PC-3细胞并表达;该包装蛋白免疫BALB/c小鼠后,小鼠血清IgG1和IgG2a抗体水平同时升高。结论:本研究研制的DNA包装蛋白GST-RGD-ΔHBcAg可作为基因治疗载体,并能对宿主的免疫系统起调节作用,为肿瘤的免疫——基因治疗载体提供一个新的选择。

Objective ： To develop a novel protein carrier which can not only regulate the immune system but also deliver DNA into the tumor ceil as an effective non-viral gene delivery system. Methods： By using gene engineering techniques, we constructed a fusion protein containing the -COOH end of human hepatitis B virus core antigen （HBcAg） , small home-to-cancer peptide ligand RGD and Glutathione S-transferase （GST） , which was expressed in E. coil and purified by size exclusion chromatography and affinity chromatog- raphy. We labeled it with FITC to observe whether it could bind prostate cancer PC-3 ceil lines, and meanwhile used it as a non-viral gene delivery carrier with the plasmid pEGFP-NI that could express GFP in PC-3 cells. Furthermore, we observed the regulatory func- tion of this fusion protein to the mouse immune system. Results ： The results of SDS-PAGE showed that the new protein carrier was obtained, which It could enter PC-3 cells with DNA in vitro and induce the mouse immune system to produce IgG1 and IgG2ct simulta- neously. Conclusion： The new protein carrier can be used as a target protein, especially in positive cells and the immune system. It promises to be a good novel carrier for the gene therapy of cancer. Natl J Androl, 2008, 14（10） ： 888-892