摘要
目的:克隆人颗粒溶素和穿孔素基因并构建真核表达载体pGNLY-2A-PFP,观察其在人肺癌A549细胞中的表达情况。方法:体外分离培养外周血单个核细胞并抽提总RNA,RT-PCR扩增人颗粒溶素和穿孔素的基因片段,并将其分别插入pMD18-T进行测序,鉴定正确后构建pIRES-GNLY、pIRES-PFP及pGNLY-2A-PFP并转染人肺癌A549细胞,RT-PCR和间接免疫荧光检测目的蛋白表达,流式细胞Annexin V/PI检测转染后细胞凋亡情况。结果:成功获取了人颗粒溶素和穿孔素cDNA,构建了真核表达载体pIRES-GNLY、pIRES-PFP、pGNLY-2A-PFP,转染A549细胞后检测出了目的蛋白的表达,pGNLY-2A-PFP转染组细胞死亡率高于其他对照组(P<0.05),死亡细胞以凋亡为主。结论:人颗粒溶素和穿孔素基因可以在人肺癌A549细胞中表达,二者共表达能够促进细胞凋亡,这将有助于颗粒溶素和穿孔素在肿瘤治疗中应用的后续研究。
Objective：To construct eukaryotic co-expression vector pGNLY-2A-PFP and to observe its expression in A549 cells.Method： Total RNA was extracted from cultured PBMC.Granulysin and perforin gene segments were obtained with specific primers by RT-PCR and then respectively inserted into pMD18-T plasmid for sequencing.After identification,eukaryotic vectors pIRES-GNLY,pIRES-PFP and pGNLY-2A-PFP were transfected into A549 cells.The expression of target genes was detected by RT-PCR and indirect immunofluorescence.Apoptosis of A549 cells was evaluated by flow cytometry（AnnexinV/PI）.Result： The eukaryotic vectors pIRES-GNLY,pIRES-PFP and pGNLY-2A-PFP were successfully constructed.Target protein expression was detected after transfection.FCM results showed that the death rate of pGNLY-2A-PFP transfected cells was markedly increased（P〈0.05）.Conclusion： Granulysin and perforin could co-express in A549 cells and could mediate apoptosis.These findings would be help for further study on the mechanism of cellular immunity and immune therapeutic effect of granulysin co-operating with perforin.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2009年第10期12-17,共6页
China Biotechnology
