颗粒溶素和穿孔素真核共表达及其对A549细胞的影响

中国生物工程杂志

2009, Vol. 29

Issue (10): 12-17

研究报告

颗粒溶素和穿孔素真核共表达及其对A549细胞的影响

王晚霞¹,兰喜²,徐向红¹,居军^1*,柳纪省²

1.甘肃省人民医院临床检验中心兰州 730000
2.中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,农业部畜禽病毒学重点开放实验室兰州 730070

Eukaryotic Co-expression of Granulysin and Perforin and Their Effects on A549 Cells

WANG Wan-xia¹,LAN Xi²,XU Xiang-hong¹,JU Jun¹,LIU Ji-xing²

1.Laboratory Center,Gansu Provincial People’s Hospital, Lanzhou 730000,China
2.State Key Laboratory of Veterinary Etiological Biology,Key Laboratory of Animal Virology of Ministry of Agriculture,Lanzhou Veterinary Research Institute,CAAS, Lanzhou 730070,China

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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。

关键词： 颗粒溶素; 穿孔素; 人肺癌A549细胞; FMDV2A

Abstract:

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 constructed and 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 (Annexin V/PI) after transfection . 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 farther study on the mechanism of cellular immunity and immune therapeutic effect of granulysin co-operating with perforin．

Key words: Granulysin Perforin Lung neoplasms FMDV2A

收稿日期: 2009-02-09 出版日期: 2009-10-29

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通讯作者: 居军 E-mail: jjwork@163.com

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引用本文:

王晚霞兰喜徐向红居军柳纪省. 颗粒溶素和穿孔素真核共表达及其对A549细胞的影响[J]. 中国生物工程杂志, 2009, 29(10): 12-17.

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https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I10/12

[1]	夏玉洁吴初新胡成钰. 草鱼穿孔素C端溶细胞活性分析[J]. 中国生物工程杂志, 2010, 30(01): 47-50.

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