介导可溶性MHCⅠ产生的去整合素金属蛋白酶初步筛选研究

Screening of ADAMs that can mediate the generation of soluble MHCⅠ

目的:以HEK293细胞为研究模型,筛选介导可溶性MHCⅠ产生的去整合素金属蛋白酶。方法:将ADAM8、ADAM10、ADAM15和ADAM17cDNA分别克隆入pcDNA3.1/V5载体,并用TransIT-LT1转染试剂将构建好的载体转染入HEK293细胞,用潮霉素B筛选ADAMs蛋白稳定表达克隆;用SDS-PAGE、免疫印迹结合化学发光法检测ADAMs在HEK293细胞中的表达;用细胞表面生物素标记、免疫沉淀、SDS-PAGE、免疫印迹结合化学发光检测ADAMs过表达对可溶性MHCⅠ产生的影响。结果:成功获得稳定表达ADAMs的HEK293细胞;过表达ADAM10和ADAM17可增强HEK293细胞产生可溶性MHCⅠ。结论:ADAM10和AD-AM17具介导可溶性MHCⅠ产生的潜能。

AIM：To screen ADAMs that can mediate the generation of soluble MHCⅠ using HEK293 cells as study model.METHODS：ADAM8,ADAM10,ADAM15 or ADAM17 cDNA expressing vectors were separately constructed with pcDNA3.1/V5 as vectors and were transfected into HEK293 cells.Hygromycin B was used to screen the cells that stably expressed ADAMs.After HEK293 cells were cell-surface labeled with biotin and cultured for another 4 h and soluble MHCⅠ the medium was immnoprecipitated with W6/32,the effect of ADAM＇s over-expression on the generation of soluble MHCⅠ was examined by SDS-PAGE,Western blot and enhanced chemiluminescence.RESULTS：HEK293 cells stably expressing ADAMs were successfully obtained and the over-expression of ADAM10 or ADAM17 increased the release of soluble MHCⅠ.CONCLUSION：ADAM10 and ADAM17 have the potentials of mediating the generation of soluble MHCⅠ.