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目的探讨白细胞介素(IL)24基因表达目的蛋白IL-24的方法,检测IL-24对肝癌细胞生长的影响。方法自经5μg/ml植物血凝素(PHA)刺激的正常人单个核细胞(PBMCs)中提取总RNA,RT-PCR扩增出IL-24cDNA,插入到pHAGE-CMV-MCS-IzsGreen中构建成慢病毒转移载体pHAGE-IL-24,转染HEK293T细胞。荧光显微镜观察293T细胞中绿色荧光蛋白(GFP)表达;梯度稀释法测定病毒滴度,感染HEK293T细胞48h后进行RT-PCR和Westernblot检测IL-24的基因和蛋白表达;MTT法检测细胞的增殖。结果限制性内切酶检测和基因测序证实成功构建了携带IL-24基因的慢病毒表达载体,滴度为5×106TU/ml。以MOI为5.0的重组慢病毒感染靶细胞HepG248h后,能够检测到外源基因IL-24的蛋白表达。IL-24能够抑制肝癌细胞的生长。结论成功构建了含IL-24基因的慢病毒表达载体,获得的病毒能够有效感染HepG2细胞,并在其中大量表达目的蛋白;IL-24能够抑制肝癌细胞的生长。
Objective To investigate the expression of interleukin (IL) 24 in IL-24 and to investigate the effect of IL-24 on the growth of hepatoma cells. Methods Total RNA was extracted from normal human mononuclear cells (PBMCs) stimulated with 5μg / ml phytohaemagglutinin (PHA). IL-24 cDNA was amplified by RT-PCR and inserted into pHAGE-CMV-MCS-IzsGreen Lentiviral transfer vector pHAGE-IL-24 was transfected into HEK293T cells. The expression of green fluorescent protein (GFP) in 293T cells was observed by fluorescence microscopy. The virus titer was determined by gradient dilution method. The expression of IL-24 gene and protein was detected by RT-PCR and Western blot 48 h after HEK293T cells infection. Cell proliferation was measured by MTT assay. Results The restriction endonuclease assay and gene sequencing confirmed that the lentiviral vector carrying IL-24 gene was successfully constructed with the titer of 5 × 106TU / ml. The target gene HepG248h was infected with recombinant lentivirus with an MOI of 5.0, and the protein expression of the exogenous gene IL-24 could be detected. IL-24 can inhibit the growth of liver cancer cells. Conclusion The recombinant lentiviral vector containing IL-24 gene was successfully constructed. The obtained virus can effectively infect HepG2 cells and express a large amount of the target protein in it. IL-24 can inhibit the growth of hepatoma cells.