鼠ICOSIg融合蛋白腺病毒的制备及其对胰岛细胞系NIT-1的感染  

Construction of recombinant adenovirus expressing mouse ICOSIg and its infection to NIT-1 islet cells

作　　者：许雪青[1] 王丰[2] 陈雪丹[1] 熊加祥[1] 段文元[1] 白云[1] 

机构地区：[1]第三军医大学医学遗传学教研室,重庆400038 [2]第三军医大学西南医院检验科,重庆400038

出　　处：《第三军医大学学报》2006年第21期2135-2137,共3页Journal of Third Military Medical University

基　　金：国家自然科学基金资助项目(30300170)~~

摘　　要：目的制备mICOSIg腺病毒,研究其对胰岛细胞系NIT-1的感染情况,及可否使后者正确表达分泌mICOSIg。方法从pMIG-ICOSIg质粒上切下ICOSIg融合蛋白的表达框,插入pAdtrack-CMV中构建腺病毒穿梭质粒pAdmICOSIg,在Adeasy-1大肠杆菌中进行同源重组,随后转染293细胞进行病毒颗粒包装。收集病毒后感染NIT-1细胞,用免疫印迹证实mICOSIg在NIT-1细胞中的正确分泌表达。结果ICOSIg编码框被正确构建到pAdtrack-CMV中,并经测序证实无误。目的病毒成功包装出来,并能有效感染胰岛细胞系NIT-1,免疫印迹证实培养上清中存在正确大小的mICOSIg融合蛋白。结论成功制备了mICOSIg腺病毒,该病毒能有效将mICOSIg转入胰岛细胞系NIT-1,并能准确分泌mICOSIg蛋白。为研究1型糖尿病中ICOS信号通路在T细胞破坏胰岛细胞中的作用提供了有效的研究手段。Objective To construct the adenovirus that expresses mouse ICOSIg （mICOSIg, mouse ICOS mouse IgG fusion protein） and study its ability to infect NIT-1 cells to secret mICOSIg. Methods The ICOSIg fragment was cut off from pMIG-ICOSIg and cloned into pAdtrack-CMV. After homologous recombination in Adeasy-1 cells, the adenovirus vector was transfected into 293 packing cells. The packed virus was harvested and used to infect NIT-1 islet cells. The secretion of mICOSIg was identified with Western blotting. Results The mICOSIg fragment was correctly inserted into pAdtrack-CMV, and the virus titer was 2.3 × 10^10. The AdmICOS virus infected NIT-1 cells well with MOI = 50, and mICOSIg was successfully secreted in infected NIT- 1 cells. Conclusion The AdmICOS adenovirus was successfully constructed and infected NIT-1 islet cells well. mICOSIg could be secreted successfully by infected NIT-1 islet cells.

关 键 词：ICOS 腺病毒 胰岛细胞 

分 类 号：R322.57[医药卫生—人体解剖和组织胚胎学] R373[医药卫生—基础医学]