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目的构建HLA-DRB1~*0901及HLA-DRA基因真核细胞双表达载体,并对其是否表达鉴定。方法用RT-PCR的方法从人外周血淋巴细胞的mRNA中逆转录扩增出HLA-DRB1~*0901和HLA-DRA的cDNA,HLA-DRA经BglⅡ酶切插入真核表达载体pVITRO2的MCS2中,同时确认正反向,再在pVITRO2的MCS1中连入BamHⅠ酶切的HLA-DRB,确认正反向。将双表达载体转染猪肾细胞,Western-blotting鉴定是否表达。结果酶切和PCR分析鉴定,得到了pVITRO2-DRA-DRB1~*0901双表达载体,Western-blotting鉴定出有表达。结论双表达载体pVITRO2可以在猪肾细胞中表达HLA-DRB1~*0901。
Objective To construct eukaryotic double-expression vector of HLA-DRB1 * 0901 and HLA-DRA gene and to identify whether it is expressed or not. Methods cDNA of HLA-DRB1 * 0901 and HLA-DRA were reverse transcribed from human peripheral blood lymphocyte mRNA by RT-PCR and inserted into MCS2 of eukaryotic expression vector pVITRO2 by BglII digestion , While confirming the forward and reverse, then BamH I digested HLA-DRB was ligated into MCS1 of pVITRO2, confirming positive and negative. The double-expression vector was transfected into porcine kidney cells, Western-blotting identification of expression. Results Double enzyme digestion and PCR analysis identified pVITRO2-DRA-DRB1 ~ * 0901 double expression vector, Western-blotting identified the expression. Conclusion Double expression vector pVITRO2 can express HLA-DRB1 ~ * 0901 in porcine kidney cells.