HLA-DR基因的克隆及其在小鼠黑色素瘤细胞中的表达

The cloning of HLA DR gene and its expression in mouse melanoma cells

目的：获得表达ＨＬＡ－ＤＲ基因的小鼠黑色素瘤细胞。方法：应用ＲＴ－ＰＣＲ技术从人Ｂ淋巴瘤细胞系Ｒａｊｉ中克隆了ＨＬＡ－ＤＲ３α和β链ｃＤＮＡ，并经测序证实。将其分别插入至真核表达载体ｐＬＸＳＮ和ｐＣＥＰ４中，用脂质体转染小鼠黑色素瘤细胞Ｂ１６，然后用Ｇ４１８和Ｈｙｇ双重筛选。结果和结论：经Ｇ４１８和Ｈｙｇ双重筛选后获得的抗性细胞经流式细胞仪检测，表明５９％的细胞表达所转染的ＨＬＡ－ＤＲ３分子。ＰＣＲ从其基因组ＤＮＡ中扩增到α和β链基因，提示ＨＬＡ－ＤＲ３ｃＤＮＡ已整合至宿主细胞的基因组ＤＮＡ中。本研究为研究异种ＭＨＣⅡ转染在肿瘤免疫中所起的作用奠定了基础。

Objective: Obtaining mouse melanoma cells expressing HLA DR genes. Methods: HLA DR3 α And β chain cDNA were cloned by RT PCR from human B lymphoma Raji cell line and confirmed by DNA sequencing. The recombinant expressing vectors were constructed by inserting the α and β chain cDNA into pCEP4/pLXSN vectors respectively. Mouse melanoma B16 was transfected with the recombinant vectors by lipofectamine and selected with G418 and Hyg. Results and Conclusion: After G418 and Hyg selecting, the HLA DR3 transfected clones were obtained. Approximately 59% of the transfected cells expressed HLA DR when assayed by FACS. It was confirmed that the HLA DR3 cDNA had been integrated into the genomic DNA of B16 DR cells by PCR analysis. Our study enables us to further investigate the role of xenogeneic MHCⅡ in tumor gene therapy.