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为获取有功能的IV型II类反式激活因子基因 (CIITA IV ) ,诱导肿瘤细胞表达MHCII类分子 ,从IFN γ刺激的THP 1细胞中以RT PCR获得CIITA IV ,将其连接到pGEMT easy载体。对所构建的pcDNA3 1 CIITA IV型表达载体进行反复测序后发现 ,所获得的CIITA IV基因存在结构变异 ,在 2 87位插入了 3个核苷酸TAG ,使 2 86 2 88位的AAG改变成为ATAGAG(2 86 2 90 ) ,并引起其他 8个座位核苷酸 (及推导的氨基酸残基 )发生改变。将表达载体转入原先不表达MHCII类分子的HeLa细胞中 ,检测到所获得的IV型CIITA变异体具有诱导人II类分子HLA DR表达的能力。空载体和CIITA IV基因导入的HeLa细胞中 ,DR阳性细胞百分率分别为 0 0 1 %和 37 6 4 %。该基因已从GenBank得到登录号 ,表明这是一个具有诱导HLA DR分子表达功能的IV型CIITA新基因。
To obtain a functional class IV transactivator gene of type IV (CIITA IV), tumor cells are induced to express MHC class II molecules, CIITA IV is obtained by RT PCR from IFN [gamma] stimulated THP 1 cells and ligated into the pGEMT easy vector . After repeated sequencing of the constructed pcDNA3 1 CIITA IV expression vector, it was found that there was a structural variation in the obtained CIITA IV gene, insertion of 3 nucleotide TAGs at position 2 87 and change of AAG at position 2 86 2 88 to ATAGAG (2 86 2 90) and caused alterations in the other 8-seat nucleotides (and deduced amino acid residues). The expression vector was transfected into HeLa cells that did not originally express MHC class II molecules and the obtained variant CIITA variant was found to have the ability to induce HLA DR expression of human class II molecules. The percentage of DR positive cells in HeLa cells transfected with empty vector and CIITA IV gene was 0 0 1% and 37 6 4%, respectively. This gene has been obtained from GenBank accession number, indicating that this is a type IV CIITA gene with induced HLA DR molecule expression of new genes.