【摘要】 目的 探讨胃癌细胞中TWIST慢病毒表达载体的构建及构建。方法 参考Genbank的人TWIST基因序列设计出1对TWIST全基因扩增引物，从人胃癌多药耐药细胞SGC7901/VCR中扩增TWIST基因，聚合酶链反应（PCR）产物线性化克隆入慢病毒表达载体GV341中，构建重组载体GV341-TWIST。将重组载体GV341-TWIST转染至人胃癌293T细胞包装病毒，免疫荧光检测293T细胞形态改变，蛋白质印迹法（Western blot）检测细胞TWIST表达水平，实时PCR法检测病毒滴度。结果 成功构建了GV341-TWIST慢病毒表达载体，经限制性酶切后出现651 bp的条带，DNA测序分析证实重组慢病毒载体GV341-TWIST的插入序列有近100%的正确率，重组慢病毒载体转染293T细胞后，Western blot检测TWIST融合蛋白为25 kD。实时PCR测定重组慢病毒载体GV341-TWIST病毒滴度2.00E+8 TU/ml。结论 本实验成功构建了人重组慢病毒载体GV341-TWIST慢病毒表达载体，在人胃癌293T细胞中高效表达了TWIST蛋白。更多还原

【Abstract】 Objective To investigate the construction of TWIST lentivirus expression vector in gastric cancer cells.Method A pair of TWIST1 whole gene amplification primers were designed with reference to the human TWIST gene sequence in Genbank. Amplification of TWIST gene from human gastric cancer multidrug-resistant cells SGC7901/VCR.The polymerase chain reaction(PCR) product was linearized and cloned into the lentiviral expression vector GV341, and the recombinant vector GV341-TWIST was constructed. The recombinant vector GV341-TWIST was transfected into human gastric cancer 293T cell packaging virus. The morphological changes of 293T cells were detected by immunofluorescence, the expression level of TWIST was detected by Western blot, and the virus titer was detected by real-time PCR. Result The GV341-TWIST lentiviral expression vector was successfully constructed. A 651 bp band was observed after restriction enzyme digestion. DNA sequencing analysis confirmed that the insert sequence of the GV341-TWIST recombinant lentiviral vector was nearly 100% correct. The recombinant lentiviral vector was transfected to package the virus in293T cells. Western blot detection of TWIST fusion protein was 25 kD. The titer of recombinant lentivirus vector GV341-TWIST was 2.00E+8 TU/ml by real-time PCR. Conclusion In this experiment, the human recombinant lentivirus vector GV341-TWIST lentivirus expression vector was successfully constructed, and the TWIST protein was highly expressed in human gastric cancer 293T cells.更多还原