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《Academic Journal of Second Military Medical University》 2015-09
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Construction of recombinant lentiviral vector harboring shRNA of ribosomal protein S3a and its effect on cell apoptosis

LI Ying-hua;WANG Jing;QIU Lei;LI Jian-zhong;HU Zhen-lin;ZHANG Jun-ping;Department of Biochemical Pharmacy,School of Pharmacy,Second Military Medical University;  
Objective To construct a recombinant lentiviral vector harboring shRNA for mouse ribosomal protein S3a(RPS3a)and to analyze its effect on cell apoptosis.Methods Four pairs of shRNA sequences targeting mouse RPS3 a mRNA were designed,and were ligated into pLLU2G-eGFP lentiviral vector.The recombinant plasmids were co-transfected with pLV/helper plasmids into 293 Tcells to package the recombinant lentivirus and the titers of the virus were determined.The lentivirus was introduced into RAW264.7cells and levels of RPS3 a mRNA and protein were detected by real-time PCR and Western blotting analysis,respectively.The apoptosis of RAW264.7cells was detected by flow cytometry assays.Results PCR and DNA sequencing analysis confirmed that the recombinant lentivirus was successfully constructed and the virus titer was 6×107-9×107 TU/mL.Results of real-time PCR showed that the silencing efficiency of Lenti-shmRPS3 awas 72.64%,and Western blotting analysis showed that RPS3 aprotein expression was decreased.Flow cytometry demonstrated that lentiviral-shRPS3 a significantly increased cell apoptosis compared with the control group(P0.05).Conclusion The constructed lentiviral vector harboring shRNA of RPS3 acan efficiently silence RPS3 agene expression and promote cell apoptosis.
【Fund】: 国家自然科学基金(91013014)~~
【CateGory Index】: R3416
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