TIPE3长型及短型过表达细胞模型的构建及鉴定

Construction and Identification of Long TIPE3 and Short TIPE3 Over-expressing Cell Models

目的:构建TIPE3长型及短型过表达慢病毒载体,转染MGC-803胃癌细胞株,筛选出稳定表达目的基因的细胞株并进行鉴定,为研究TIPE3长型及短型异构体对胃癌细胞生物学功能的影响提供重要的实验细胞模型。方法:检索NCBI基因库中人TIPE3长型及短型基因序列并设计相应引物,PCR扩增目的基因;将GV341载体和目的基因分别进行双酶切后重组,病毒包装后感染MGC-803胃癌细胞系,嘌呤霉素筛选得到稳定表达目的基因的细胞,RT-qPCR和Western blot分别从mRNA和蛋白水平对目的基因的过表达效果进行鉴定。结果:基因测序结果表明重组过表达慢病毒载体中Long TIPE3、Short TIPE3与NCBI中检索到的基因序列一致;倒置显微镜观察各组细胞形态基本一致,表明病毒转染并未影响细胞的生长;RT-qPCR和Western blot结果表明,以空病毒转染组为对照,Long TIPE3和Short TIPE3转染组中目的基因的mRNA和蛋白水平显著提高(P<0.000 1)。结论:TIPE3长型及短型过表达细胞模型构建成功,可以用来研究TIPE3长型及短型异构体对胃癌细胞生物学功能的影响。

Objective:To construct long TIPE3 and short TIPE3 over-expressing lentiviral vector,transfect MGC-803 gastric cell line,and then screen and identify the cells stably expressing the target gene,so as to provide an important experimental cell model for studying the effects of long TIPE3 and short TIPE3 on the biological function of gastric cancer cells.Methods:The human long and short TIPE3 gene sequences in the NCBI bank were searched and the corresponding primers were designed to amplify the target gene by PCR.The GV341 vector and the target gene were double-digested and recombined respectively,and the recombined lentiviral vector was packaged to infect the MGC-803 gastric cancer cell line.After puromycin screening,the cells stably expressing the target gene were obtained.The mRNA and protein levels of over-expressing target gene were identified by RT-PCR and Western blot.Results:The gene sequencing indicated that the sequences of long TIPE3 and short TIPE3 in the recombinant lentiviral vector were consistent with those in NCBI.The morphology of the cells in each group was basically the same under an inverted microscope,indicating that virus transfection did not affect the cell growth.The empty vector group was used as the control,RT-qPCR and Western blot showed that the mRNA and protein levels of the target gene in long TIPE and short TIPE3 transfected groups were significantly increased(P<0.000 1).Conclusion:The long TIPE3 and short TIPE3 over-expressing cell models are successfully constructed,and can be used to study the effects of long TIPE3 and short TIPE3 on the biological function of gastric cancer cells.