摘要
目的基于Gateway技术构建人转化生长因子诱导蛋白(human Transforming Growth Factor-induced protein,hTGFBI)高表达的人间皮瘤细胞株H2596,为后续间皮瘤增敏研究提供基础。方法商业购买hTGFBI全长基因原核细胞质粒,采用常规分子克隆技术及Gateway技术构建其慢病毒表达载体pDEST-hTGFBI并测序鉴定;pDEST-hTGFBI转染293T细胞进行慢病毒包装后感染人间皮瘤H2596细胞株;采用RT-qPCR和Western Blot分别从mRNA水平及蛋白质水平检测感染前后hTGFBI在H2596细胞株中的表达情况。结果测序结果证实成功构建慢病毒表达载体pDEST-hTGFBI;RT-qPCR及Western Blot结果提示转染后,H2596细胞株中hTGFBI mRNA及蛋白质平均表达水平分别为对照组的9.98和19.2倍,显著升高(P<0.05)。结论成功构建hTGFBI慢病毒表达载体pDEST-hTGFBI,并且hTGFBI在人间皮瘤H2596细胞株中呈高表达。
Objective To construct mesothelioma cell line H2596 with high expression of human Transforming Growth Factor-induced protein(hTGFBI) by Gateway technology so as to lay a foundation for further study on malignant mesothelioma sensitization. Methods Full-length prokaryotic plasmid of hTGFBI was bought from commercial biotech company. The hTGFBI lentiviral vector(p DEST-hTGFBI) was constructed by conventional molecular cloning and Gateway technology, and then was identified by PCR and gene sequencing. The pDEST-hTGFBI, which were added to transfect 293 T cells,conducted by Lentiviral Packaging, and transfected into H2596 cells. Real Time PCR and Western blotting analysis were individually carried out to confirm the expression of hTGFBI in H2596 cells in mRNA and protein level before and after infection. Results The hTGFBI lentiviral vector pDEST-hTGFBI was constructed successfully as identified by PCR and gene sequencing. According to the results of Real Time PCR and Western blotting analysis, the expression of TGFBI in mRNA and protein levels in H2596 cells were respectively 9.98 times and 19.2 times higher than that of control group, the level increased remarkably(P<0.05). Conclusion The hTGFBI lentiviral vector(pDEST-hTGFBI) successfully constructed by Gateway technology and the hTGFBI shows high expression in malignant mesothelioma cell lines H2596.
作者
郑晋南
张勤
ZHENG Jinnan;ZHANG Qin(West China School of Public Healthy and West China Fourth Hospital,Sichuan University,Sichuan University,Chengdu 610041,Sichuan Province,China)
出处
《预防医学情报杂志》
CAS
2020年第9期1201-1206,共6页
Journal of Preventive Medicine Information
基金
四川省卫生和计划生育委员会科研课题2016年普及应用项目(项目编号:16PJ260)。
