摘要
目的 克隆人类白细胞抗原 G(Humanleukocyteantligen G ,HLA G)突变体cDNA ,并使其在HLA Ⅰ类阴性的K5 6 2细胞上获得稳定表达 ,为研究配 受体之间的识别机制奠定基础。方法 用RT PCR方法从人子宫蜕膜组织扩增出HLA GcDNA ,得到全长HLA GPCR产物后 ,用桥式PCR方法进行定点点突变 ,将突变的目的基因亚克隆于逆转录 ,将突变的目的基因亚克隆于逆转录mG pLNCX表达载体 ,采用感染的方法将重组质粒转入K5 6 2细胞 ,最后经G4 18筛选及有限稀释 ,利用单克隆抗体W6 32进行FACS及mRNA检测 ,观察HLA G突变体在靶细胞表面的表达。结果 HLA G突变体分子在经mG pLNCX转染的靶细胞表面获得稳定高表达。结论 成功构建了mG pLNCX表达载体 ,并使HLA G突变体分子在HLA Ⅰ类阴性的靶细胞K5 6 2细胞上获得稳定表达。
ObjectiveTo obtain human HLA G mutant cDNA and stable e xpression on HLA class Ⅰ negative K562 cells. Methods RT P CR technique was used to amplify HLA G cDNA from human decidual tissue and the full length HLA G cDNA was obtained, and the product was point mutated by a pai r of specific primer, the mutant cDNA was subcloned into retrovirus vector pLNCX and the combined plasmid named mG pLNCX. Using infection technique to transduct the recombined plasmid into the target cells followed by screening with G418 and li miting dilution; Finally, flow cytometry was adopted to detect HLA G mutant exp ression on the cells.Results HLA G mutant molecule was success fully expressed on K562 cells transducted with mG pLNCX and no expression of H LA G on the control cell transducted with plasmid pLNCX.Conclusions the vector was contructed successfully and the HLA G mutant was expressed stably on the K562 cells.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2003年第1期7-10,共4页
Immunological Journal
基金
国家自然科学基金 (30 0 70 784 )
上海市科委重点项目 (0 2DJ14 0 5 4 )资助项目
