论文部分内容阅读
目的建立NK细胞受体NKG2D真核表达载体,通过转染NK细胞系YT,初步探讨NKG2D分子对YT细胞系杀伤功能的增强作用。方法用RT-PCR方法从NK-92细胞中调取NKG2D基因片段,克隆到pGEM-T Easy载体并对克隆的DNA片段进行序列分析。用限制内切酶EcoRⅠ和BamHⅠ消化pGEM-T Easy/NKG2D重组质粒,分离NKG2D片段,并插入真核表达质粒pEGFP-N1的相应限制酶位点,酶谱分析鉴定重组表达载体pEGFP-N1/NKG2D。然后经脂质体介导转染CHO细胞和YT细胞。应用荧光显微镜观测、Western blot方法和免疫组化染色对转染细胞内pEGFP-N1/NKG2D的表达进行鉴定,MTT方法观察YT细胞对肿瘤细胞的杀伤功能。结果RT-PCR扩增获得650bp基因片段,经DNA序列分析证明所获得的DNA序列与文献报道的NKG2D序列一致。转染的CHO细胞在荧光显微镜下发出强绿色荧光,Western blot分析显示重组蛋白能特异地与抗人NKG2D单克隆抗体结合;免疫组化检测显示,转染的CHO中有棕色颗粒,证明所构建的NKG2D真核表达载体可以在细胞中表达;转染NKG2D真核表达载体的YT细胞对乳腺癌细胞具有更强的杀伤效果。结论所获得的表达NKG2D分子的真核表达载体,通过转染YT细胞,初步鉴定所表达NKG2D分子具有生物学功能,可以提高NK细胞对肿瘤细胞的杀伤活性。
Objective To establish NKG2D eukaryotic expression vector of NK cell receptor, and to investigate the enhancing effect of NKG2D on the cytotoxicity of YT cell line by transfecting NK cell line YT. Methods The NKG2D gene fragment was retrieved from NK-92 cells by RT-PCR and cloned into pGEM-T Easy vector. The cloned DNA fragment was sequenced. The recombinant plasmid pGEM-T Easy / NKG2D was digested with restriction endonucleases EcoRI and BamHI and the NKG2D fragment was isolated and inserted into the corresponding restriction sites of the eukaryotic expression plasmid pEGFP-N1. The recombinant expression vector pEGFP-N1 / NKG2D . Then transfected CHO cells and YT cells by liposomes. The expression of pEGFP-N1 / NKG2D in transfected cells was identified by fluorescence microscopy, Western blot and immunohistochemistry. The cytotoxicity of YT cells to tumor cells was observed by MTT assay. Results The 650bp gene fragment was amplified by RT-PCR. DNA sequence analysis showed that the obtained DNA sequence was consistent with the reported NKG2D sequence. The transfected CHO cells fluoresced strongly green under a fluorescence microscope and Western blot analysis showed that the recombinant protein specifically binds to the anti-human NKG2D monoclonal antibody; immunohistochemistry revealed brown particles in the transfected CHO, demonstrating that the constructed Of NKG2D eukaryotic expression vector can be expressed in cells; YT cells transfected with NKG2D eukaryotic expression vector have a stronger killing effect on breast cancer cells. CONCLUSION: The eukaryotic expression vector expressing NKG2D molecule can effectively enhance the cytotoxic activity of NK cells to tumor cells by transfecting YT cells. The preliminary identification of the expressed NKG2D molecule has the biological function.