摘要
目的克隆 HL A- E c DNA,并使其在 HL A 类阴性的靶细胞 L c L72 1.2 2 1细胞上获得稳定表达。方法用 RT-PCR方法从人外周血淋巴细胞扩增出 HL A - E c DNA ,并通过内核糖体进入位点 (IRES)将目的基因亚克隆于已经载有 HL A-A2的逆转录病毒表达载体 p GCEN上 ,构建成 HL A - A 2 / E多顺反子表达载体 (p G/ A2 E) ,采用感染的方法将重组质粒转入L c L 72 1.2 2 1细胞 ,最后经 G418筛选及有限稀释 ,利用抗 HL A- E特异的单克隆抗体 3D12进行 FACS检测 ,以观察 HL A - E分子在靶细胞表面的表达情况。结果 HL A- E分子在经 p G/ A2 E转染的靶细胞表面获得明显的表达 (88.79% ) ,且显著高于表达 HL A- E的对照细胞株 JAR(2 6 .2 1% ) ,而经 p G/ A2载体转染的靶细胞则未获得表达。结论成功构建了 p G/ A2 E多顺反子表达载体 ,并使 HL A- E分子在 HL A 类阴性的 L c L72 1.2 2
ObjectiveTo clone human HLA E cDNA and obtain stable expression on HLA classⅠnegative LcL721 221 cell Methods RT PCR technique was employed to amplify HLA E cDNA from human PBMC, the cDNA was subcloned into retrovirus vector pGCEN carrying HLA A2 cDNA through internal ribozyme entry site (IRES) thus the multi cistron expression vector was constructed and named pG/A2E Then infectious method was used to transduct the recombinant plasmid into the target cells followed by screening with G418 and limiting dilution; Finally, flow cytometry was adopted to detect HLA E expression on the target cells Results HLA E molecules were successfully expressed on LcL721 221 cells tranducted with pG/A2E (88 79%) and the expression was higher than control JAR (26 21%), but the expression of HLA E molecules was not detected on LcL721 221 cells transducted with pG/A2 Conclusion The multi cistron expression vector was constructed successfully and the HLA E molecules were expressed on LcL721 221 cells
出处
《免疫学杂志》
CAS
CSCD
北大核心
2001年第6期403-405,424,共4页
Immunological Journal
基金
国家自然科学基金资助项目 (30 0 70 784)
