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| 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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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．
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Received: 09 February 2009
Published: 29 October 2009
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