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目的:构建金黄色葡萄球菌肠毒素C3(SEC3)过表达慢病毒载体并体外检测其表达目的基因。方法:聚合酶链反应(PCR)扩增SEC3基因片段;用Age I酶切线性化GV365慢病毒载体,通过连接反应构建GV365-SEC3载体,运用PCR方法鉴定阳性克隆载体;转染293T细胞包装慢病毒,观察细胞荧光及Western blot检测慢病毒载体表达,HIV-1 p24 ELISA法测定慢病毒载体滴度。结果:获得目的基因,并成功构建GV365-SEC3慢病毒载体,通过PCR及DNA测序鉴定,证明GV365-SEC3质粒构建正确;转染293T细胞后可观察到大量荧光细胞,经蛋白电泳得到29 k D大小的蛋白条带,与目的基因蛋白相符合,ELISA检测病毒载体滴度为5×10~8TU/ml。结论:成功构建GV365-SEC3过表达慢病毒载体,为后期研究其体内外对抗肿瘤的作用与机制奠定基础。
Objective: To construct the lentiviral vector of staphylococcal enterotoxin C3 (SEC3) overexpression and to detect the expression of the target gene in vitro. Methods: The SEC3 gene fragment was amplified by polymerase chain reaction (PCR). The GV365 lentiviral vector was digested with Age I, and the GV365-SEC3 vector was constructed by ligation reaction. The positive cloning vector was identified by PCR. The expression of lentiviral vector was detected by fluorescence and Western blotting. The titer of lentiviral vector was determined by HIV-1 p24 ELISA. Results: The target gene was obtained and the lentiviral vector GV365-SEC3 was successfully constructed. The recombinant plasmid was verified by PCR and DNA sequencing. The recombinant plasmids of GV365-SEC3 were constructed correctly. A large number of fluorescent cells were observed after transfection with 293T cells. The size of the protein band, in line with the target gene protein, ELISA detection of viral vector titer of 5 × 10 ~ 8TU / ml. Conclusion: The construction of GV365-SEC3 overexpression lentiviral vector lays the foundation for the later study of its anti-tumor effect in vitro and in vivo.