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目的构建可以表达人类HLA-DRB1*0803的转基因载体,并在细胞水平验证其表达及抗原递呈功能。方法提取人WATANABE细胞株(基因型为HLA-DRB1*0803纯合子)的总RNA,RTPCR扩增DRB1*0803基因片段,与小鼠Eαpromoter和兔β-globin基因内含子序列同时导入p BR002-lacz载体。pBR002-HLA-DRB1*0803转基因载体转染小鼠DCS细胞和B细胞系,用Western blot和免疫荧光的方法对HLA-DRB1蛋白进行检测。流式细胞术检测转染小鼠DCS细胞对HBc Ag特异性CD4+T细胞的刺激和极化效应。结果经双酶切和测序验证,成功构建了p BR002-HLA-DRB1*0803转基因载体;转染载体后的小鼠DCS细胞和B细胞系均表达出了大小约为29×10~3的HLA-DRB1蛋白;免疫荧光结果表明HLA-DRB1蛋白主要表达于细胞质内。转染后的小鼠DCS细胞可将负载的HBcAg递呈给HLA-DRB1*0803阳性基因型的人CD4~+T细胞,产生明显的CD4~+T细胞应答。结论成功构建了HLA-DRB1*0803转基因载体,该载体可以在细胞水平特异性表达HLA-DRB1蛋白,并具有HLADRB1*0803特异性抗原递呈功能。
Objective To construct a transgenic vector that can express human HLA-DRB1 * 0803 and verify its expression and antigen presenting function at the cellular level. Methods The total RNA of human WATANABE cell line (genotype HLA-DRB1 * 0803 homozygote) was extracted. The DRB1 * 0803 gene fragment was amplified by RTPCR and inserted into the pBR002- lacz vector. The pBR002-HLA-DRB1 * 0803 transgenic vector was transfected into mouse DCS cells and B cell line, and the HLA-DRB1 protein was detected by Western blot and immunofluorescence. Flow cytometry was used to detect the stimulation and polarization of HBcAg-specific CD4 + T cells transfected mouse DCS cells. Results The pBR002-HLA-DRB1 * 0803 transgene vector was successfully constructed by double enzyme digestion and sequencing. The expression of HLA-DRB1 * 0803 gene was detected in both DCS and B cell lines after transfection -DRB1 protein; immunofluorescence results show that the HLA-DRB1 protein is mainly expressed in the cytoplasm. The transfected mouse DCS cells can deliver the loaded HBcAg to human CD4 ~ + T cells of HLA-DRB1 * 0803 positive genotype and produce a significant CD4 ~ + T cell response. Conclusion The HLA-DRB1 * 0803 transgenic vector was successfully constructed. The vector can express HLA-DRB1 protein at the cellular level and has the antigen presenting function of HLADRB1 * 0803.