IV型II类反式激活因子基因新变异体的克隆、鉴定和功能测定

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。

Cloning of a Type IV CIITA Gene Variant and Determination of Its Biological Function for Induction of HLA-DR Expression

To test the ability of type IV class II transactivator (CIITA-IV) gene for induction of MHC class II expression on tumor cells, the gene was amplified by PCR from cDNA of the IFN-γ-activated THP1 cell line and cloned into plasmid pGEMT-easy. The CIITA-IV gene was then removed from the constructed plasmid pGEMT-CIITA-IV and ligated into pcDNA3.1 to construct the expression vector pcDNA3.1-CIITA-IV. Quite unexpectedly, our repeated sequencing for the CIITA-IV indicated that there were three new nucleotides inserted at position 287 to change the sequences from AAG (286-288) to ATAGAG (286-290). Besides, there took place eight nucleotides replacements at different position, resulting in substitutions of eight deduced amino acids. For functional assay, the CIITA-IV-containing expression vector was transfected into HeLa cells that failed to express class II molecules. Our cloned CIITA-IV gene variant was able to work in the tumor cells for induction of class II expression as determined by flow cytometry. The percentages of the HLA-DR-positive HeLa cells were increased from 0.01% to 37.64% after the transfection. The CIITA-IV was thus capable of having its accessible number from GenBank as a new genetic entity.