Journal of Jilin University Medicine Edition ›› 2016, Vol. 42 ›› Issue (03): 452-456.doi: 10.13481/j.1671-587x.20160307

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Construction of IRF5 gene lentiviral vector and its expression in macrophages RAW264.7

MAI Li1, WEI Jianrui1, HUA Xing2   

  1. 1. Department of Cardiology, Affiliated Guangzhou Red Cross Hospital, College of MedicalSciences, Jinan University, Guangzhou 510515, China;
    2. Department of Pathology, Affiliated Guangzhou Red Cross Hospital, College of Medical Sciences, Jinan University, Guangzhou 510515, China
  • Received:2015-11-16 Published:2016-06-17

Abstract:

Objective: To construct interferon regulatory factor 5(IRF5) gene lentiviral vector LV11-cmv-neo-IRF5 and transfect it into macrophages RAW264.7, and to observe its expression in RAW264.7 cells. Methods: The target gene IRF5 gragment was fished by PCR and cloned into linearized lentiviral vector LV11 by using DNA recombinant technique. The recombinant vector was identified by PCR, double enzyme digestion,and sequencing and the 293T cells were transfected with the lipofectin reagent for lentiviral particles packaged with expression and packing plasmids. The supernatant of lentiviral was collected and infected into the macrophages RAW264.7. After screening the cells, which were not infected with G418, the positive infected cells(RAW264.7-IRF5)were obtained (experiment group). Meanwhile, the RAW264.7-NC cells was regarded as controls(control group).The expression levels of IRF5 mRNA and protein in RAW264.7 cells in two groups were examined by RT-qPCR and Western blotting methods. Results: The PCR and enzyme digestion analysis results confirmed that the lentiviral vector LV11-cmv-neo-IRF5 was successfully constructed. The RAW264.7 cells were infected with virus supernatant and the positive cells were obtained by G418 screening.The RT-qPCR results showed that compared with control group, the expression level of IRF5 mRNA in the RAW264.7 cells in experiment group was increased (P<0.01);the Western blotting results showed that compared with control group,the IRF5 protein epression level in the RAW264.7 cells in experiment group was increased. Conclusion: The lentiviral vector LV11-cmv-neo-IRF5 containing IRF5 gene is successfully constructed,and the IRF5 gene can express stably in the macrophages RAW264.7.

Key words: interferon regulatory factor 5, lentiviral vector, atherosclerosis, macrophages

CLC Number: 

  • Q78