靶向融合防龋DNA疫苗的构建与体外细胞表达研究  

Construction and expression in vitro of a targeted fusion anticaries DNA vaccine

作　　者：郭继华[1] 樊明文[1] 边专[1] 贾荣[1] 陈智[1] 彭彬[1] 

机构地区：[1]武汉大学口腔医学院口腔生物医学工程教育部重点实验室,430079

出　　处：《中华口腔医学杂志》2003年第4期282-284,共3页Chinese Journal of Stomatology

摘　　要：目的　构建以人细胞毒性T淋巴细胞抗原 4 (cytotoxicTlymphocyte associatedantigen4 ,CTLA4 )为引导的抗变形链球菌 (Streptococcusmutans,S mutans)葡糖基转移酶(glucosyltransferase ,GTFs)Ⅰ和表面蛋白PAc的靶向融合防龋DNA疫苗pGJA P ,检测其在真核细胞中的表达。方法　利用RT PCR方法从外周淋巴细胞中获得CTLA4胞外区和Igγ1 恒定区基因并进行T A克隆 ,获得携带CTLA4 Ig融合基因的重组质粒 pGJ,选择合适的酶切位点 ,再克隆入融合防龋DNA疫苗 pGLUA P中 ,构建靶向融合防龋DNA疫苗pGJA P ,并转染CHO细胞系 ,用蛋白质免疫印迹实验检测其表达。结果　重组质粒 pGJ、pGJA P分别含有CTLA4 Igγ1 融合基因和CTLA4 、Igγ1 、pac、glu基因序列。蛋白质免疫印迹结果显示 ,pGJA P表达的抗原可以与特异性抗PAc抗体发生免疫反应。结论　成功构建了靶向融合防龋DNA疫苗 pGJA P 。Objective To construct and detect the targeted anti caries fusion DNA vaccine pGJA P encoding the A P fragment of pac, glu fragment of gtfB and extracellular region of the human CTLA4 and the Fc region of human Igγ 1 gene for the targeting antigen to APC Methods The extracellular region of the human CTLA4 and the Fc region of human Igγ 1 genes were amplified by RT PCR from human lymphocytes, and the genes were cloned into pUC m T vector respectively After sequencing, Fc region of human Igγ 1 gene was cloned to the downstream of CTLA4 gene fragment as the recombinant plasmid pGJ The fusion gene was then inserted into the plasmid pGLUA P to get the recombinant plasmid pGJA P The CHO cells were transfected with liposome and the expression of fusion protein in cultured supernatants were detected using Western blotting Results The plasmids pGJ and pGJA P carried the CTLA4 Ig fusion gene and CTLA4 Ig fusion gene, A P fragment of pac gene and glu fragment of gtfB gene respectively The expressed protein could response to specific anti PAc antibody Conclusion The targeted fusion anti caries DNA vaccine pGJA P is constructed successfully and expressed in eukaryotic cells correctly

关 键 词：靶向融合 防龋DNA疫苗 体外细胞表达 细胞毒性 T淋巴细胞抗原4 CTLA4 链球菌 

分 类 号：R780.1[医药卫生—口腔医学]