摘要
目的:构建载有小鼠CD40-IgG2aFc-IRS-GFP融合基因片段的腺病毒载体,并检测其转染293细胞后的融合蛋白表达及出毒情况。方法:提取小鼠CD40、IgG2aFc基因片段构建PDC316-IgG2aFc-IRS-GFP质粒,测序成功后包装构建Ad5-PDC316-CD40-IgG2aFc-IRS-GFP,转染293细胞。出毒后大量复制病毒并纯化,测定病毒滴度,体外感染细胞观察病毒复制及分泌目的蛋白情况并检测目的蛋白表达量。结果:成功构建了质粒及病毒,体外感染显示该病毒能有效地感染细胞并表达蛋白(荧光显示),检测目的蛋白表达量随时间点及浓度增加而增加。当病毒浓度增加到一定程度时目的蛋白表达趋于无明显差异性。结论:构建小鼠融合基因片段并成功转入细胞内、分泌表达目的蛋白是可行的,为进一步研究该病毒在大鼠体内感染及表达CD40Ig、大鼠肝移植模型免疫耐受及其机制的研究奠定基础。
Objective To construct an adenovirus vector containing mouse CD40-IgG2aFc-IRS-GFP fusion gene, and detect the expression of the fusion protein in the transfected 293 cells. Methods The fragments of CD40 and IgG2aFc were extracted and then inserted into plasmid PDC316-IgG2aFc-GFP. After being identified by sequencing, Ad5-PDC316- CD40-IgG2aFc-GFP was transfected into 293 cells. The virus titer was measured after massive viral replication and purification ; the expression of the fusion protein was analyzed. Results The plasmid and adenovirus were successfully constructed. The virus effectively transfected 293 cells in which the fusion protein was expressed with fluorescence, and the expression level increased with time and the increasing viral concentration. The protein expression, however, did not differ significantly after the viral concentration reached a certain level. Conclusions The mouse fragments of fusion genes has been constructed and successfully transferred to the cells in which the fusion protein is expressed. This study lays the foundation for further research on adenovirus infection in mice and on immune tolerance in the murine model of liver transplantation and its mechanisms.
出处
《实用医学杂志》
CAS
北大核心
2009年第13期2041-2044,共4页
The Journal of Practical Medicine
基金
浙江省自然科学基金资助项目(编号:X204003)
