|Alternative Title||Expression of Human Granulysin and Perforin and their Effects on Lung neoplasms
|Place of Conferral||兰州
3. 以FMDV 2A代替IRES，构建真核共表达载体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。重组质粒转染A549细胞后检测出了目的基因mRNA及蛋白的表达，pGNLY-2A-PFP转染后细胞死亡率高于其他对照组（P<0.05），死亡细胞以凋亡为主。
|Other Abstract||Granulysin and perforin are cytolytic protein presented in the granules of human cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells, which exhibit cytolytic activity against a broad range of microbes and tumors.Granulysin and perforin are expected to be ideal drugs in the use of anti-tumor and anti-tuberculosis.Granulysin and perforin are implicated in a myriad of diseases including infection, cancer, transplantation. Researches of granulysin and perforin will be very helpful to diagnosis and treatment of many diseases. In this study, granulysin and perforin gene segments were cloned from cultured PBMC. Different molecular weight granulysin fusion protein were recombinanted using prokaryotic expression system. Co-expression eukaryotic vector of granulysin and perforin was constructed and transfected into A549 cells. Effects which granulysin and perforin co-operated in A549 cells were observed after transfection.
The methods and results were as follow.
1. Total RNA was extracted from cultured PBMC. Granulysin and perforin gene segements were obtained with specific primers by RT-PCR and then respectively inserted into pMD18-T plasmid for sequencing. Sequencing results showed that the cloned granulysin and perforin gene segements were in the same with the sequences in Genbank.Vectors pMD-GNLY and pMD-PFP were successfully constructed.
2. To construct prokaryotic expression vectors pET-GNLY9K and pET-GNLY15K and transfer them into E. coli Rosetta (DE3).Fusion protein were expressed under the induction of IPTG.SDS-PAGE and Western-blotting showed that the corresponding molecular weight of 31kD and 37kD granulysin fusion protein，which had satisfactory antigen-antibody reaction with anti-granulysin antibody, were highly expressed in E. coli after induction.
3. To construct co-expression eukaryotic vector pGNLY-2A-PFP and transfect them into A549 cells. The expression of target genes was detected by RT-PCR and indirect immunofluorescence. 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).
1. Granulysin and perforin gene segements were successfully obtained in vitro, which would be helpful for the further studies of granulysin and perforin.
2. Different molecular weight granulysin were successfully expressed using prokaryotic expression system, which would be helpful for the further study of granulysin.
3. Granulysin and perfo...|
王晚霞. 颗粒溶素与穿孔素的表达及其对肺癌细胞影响的研究[D]. 兰州. 兰州大学,2009.
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