人FAM172A干扰慢病毒载体的构建及在甲状腺乳头状癌细胞中的表达

Construction of Interfering Lentiviral Vector of Human FAM172A and its Expression in Human Papillary Thyroid Carcinoma Cells

目的构建人FAM172A干扰慢病毒表达载体,并包装成FAM172A干扰慢病毒感染甲状腺乳头状癌细胞予以鉴定。方法根据人FAM172AmRNA序列设计退火Oligo,与慢病毒载体pL/shRNA/F经酶切、连接、转化、测序鉴定。病毒包装后,转染HEK293细胞测定病毒滴度。将重组慢病毒感染甲状腺乳头状癌细胞,利用荧光显微镜和QPCR检测FAM172A的表达。结果重组慢病毒载体pL/shRNA/F-ShFAM172A经PCR扩增及测序鉴定正确,病毒包装后荧光显微镜观察到FT293细胞中有大量绿色荧光,测定病毒滴度为5×10~6TU/ml。FAM172A干扰慢病毒感染的甲状腺乳头状癌细胞中FAM172A表达被显著减少。结论成功制备了人重组FAM172A干扰慢病毒,并在甲状腺乳头状癌细胞中高效减少FAM172A的表达,为进行FAM172A的功能和机制奠定了良好的基础。

Objective To construct the interfering lentiviral expression vector of human FAM172A,package corresponding virus and identify its expression in human papillary thyroid carcinoma(PTC)cells.Methods Oligo was designed based on human FAM172A mR-NA sequence,and then digested,connected,transformed and sequenced with lentiviral vector pL/shRNA/F.After viral packaging,the HEK293 cells were transfected with recombinant lentiviral vector to detect the titer of virus.Final/y,the expression of FAM172A of PTC cells infected with recombinant lentiviral vector was analyzed by using fluorescence microscope and quantitative PCR.Results P CR and DNA sequencing assays demonstrated that the recombinant lentiviral vector of pL/shRNA/F-shFAM172A was successfully constructed. The high expression of green fluorescence protein in FT293 cells was found under fluorescent microscope.Viral titer was 5×10^6TU/ml. The level of FAM172A expression in PTC cells was significantly decreased after infection with FAM172A interfering lentivirus.Conclusion Human recombinant FAM172A interfering lentiviral vector was successfully constructed,which resulted in low FAM172A expression in PTC cells.Our results lay a good foundation to explore the functions and mechanisms of FAM172A.