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目的:构建抑癌基因WTX慢病毒载体,建立稳定转导WTX的结肠癌Lovo/WTX-EGFP细胞株,为研究WTX在结肠癌中的作用机制提供有效工具。方法:通过Gateway技术构建WTX慢病毒载体pLV.Ex3d.null-EF1A>WTX>IRES/EGFP并通过菌落PCR筛选鉴定,将其与辅助质粒pLV/helper-SL3,PLV/helper-SL4,PLV/helper-SL5共转染293FT细胞包装慢病毒并在荧光显微镜下行滴度值测定。用WTX慢病毒载体转导结肠癌Lovo细胞株,并通过多次挑克隆培养建立稳定表达WTX的Lovo/WTX-EGFP细胞株。结果:Gateway技术构建的慢病毒载体pLV.Ex3d.null-EF1A>WTX>IRES/EGFP经鉴定完全正确;慢病毒包装48 h后视野下可见清晰绿色荧光表达,病毒滴度为5×107TU/mL。慢病毒载体成功转导Lovo细胞,经qPCR及Western blot检测WTX表达水平明显升高;通过多次挑克隆培养成功建立了稳定转导WTX的Lovo/WTX-EGFP细胞株。结论:通过Gateway技术可成功构建WTX慢病毒载体并获稳定转导WTX的Lovo/WTX-EGFP细胞株,为研究WTX在结肠癌中的作用机制提供了实验基础。
OBJECTIVE: To construct WTX lentiviral vector and establish stable Lovo / WTX-EGFP cell line transfected with WTX to provide an effective tool for studying the mechanism of WTX in colon cancer. Methods: The WTX lentiviral vector pLV.Ex3d.null-EF1A> WTX> IRES / EGFP was constructed by Gateway technique and screened by colony PCR and identified with helper plasmids pLV / helper-SL3, PLV / helper-SL4, PLV / helper -SL5 cotransfected 293FT cells packaged lentivirus and measured by fluorescence microscopy titers. The colon cancer Lovo cell line was transduced with the WTX lentiviral vector and the Lovo / WTX-EGFP cell line stably expressing WTX was established by multiple pick cloning. Results: The lentiviral vector pLV.Ex3d.null-EF1A> WTX> IRES / EGFP constructed by Gateway was completely correct. The clear green fluorescence was observed in the visual field 48 h after lentivirus packaging. The virus titer was 5 × 107 TU / mL . The lentiviral vector was successfully transduced into Lovo cells, and the expression of WTX was detected by qPCR and Western blot. The Lovo / WTX-EGFP cell line stably transfected with WTX was successfully established by multiple clonal culture. CONCLUSION: The WTX lentiviral vector can be successfully constructed by Gateway technology and the Lovo / WTX-EGFP cell line stably transfected with WTX can provide an experimental basis for studying the mechanism of WTX in colon cancer.