Gateway技术构建人RhoD慢病毒表达载体及转染人黑素瘤A375细胞株

Construction and Identification of RhoD Protein Overexpressing A375 Cell Lines Via Lentiviral Vectors

目的构建人RhoD基因的慢病毒载体并进行慢病毒包装和鉴定，转染人黑素瘤细胞A375，使其过表达RhoD蛋白，为后续研究RhoD在黑素瘤中的作用奠定基础。方法Gateway技术构建携带增强型绿色荧光蛋白EGFP的RhoD慢病毒载体，经PCR及基因测序鉴定后，与辅助质粒pLV／helper-SL3，pLV／helper—SIA及pLV／helper—SL5混合采用脂质体法制备DNA-Lipofectamine2000复合物，并共同转染293FT细胞进行慢病毒包装，产生相应慢病毒颗粒，通过定量PCR方法测定病毒滴度。包装好的慢病毒转染人黑素瘤A375细胞，荧光显微镜下观察荧光表达情况，流式细胞仪检测转染效率；实验分为A375（未处理对照组）、A375-EGFP（不含目的基因的空病毒对照组）和A375-RhoD（含RhoD基因的病毒组）三组，采用实时荧光定量PCR（QPCR）及免疫印记法（western blotting，WB）验证耽扣在A375-RhoD组细胞中的过表达。结果通过PCR、基因测序证实，慢病毒表达载体pLV[Exp]-EGFP／Neo—CMV〉hRhoD构建成功。与辅助质粒共转染293FT细胞包装出具高效感染力的慢病毒，经测定病毒滴度为（5．13±2）×10^8TU／mL。转染A375细胞后可见明显的绿色荧光表达，流式检测转染效率大于80％;A375-RhoD组RhoDmRNA及蛋白水平均较A375-EGFP组和A375组细胞中明显增高。结论成功构建RhoD慢病毒表达载体，包装出具高效感染力的慢病毒颗粒并成功转染人黑素瘤A375细胞，为进一步研究RhoD在黑素瘤中的作用提供实验基础。

Objective To construct a lentiviral vector carrying human RhoD gene and to package and identify a virus particles, and transfect it into melanoma A375 cells so as to lay a foundation for further study on the role of RhoD in malignant melanoma. Methods The RhoD lentiviral vector （pLV [ Exp ]-EGFP/Neo-CMV 〉 hRhoD）was constructed by Gateway technology, and identified by PCR and gene sequencing. The lentiviral vector was mixed with helper vector pLV/helper-SL3, pLV/helper-SL4 and pLV/helper-SL5 by Lipofectamine 2 000 to prepare DNA-Lipofectamine 2 000 complexes. Then the complexes were added to transfect 293FT cells and package virus. The virus titers were determined by Quantitative PCR. The lentiviruses were transfected into A375. The expression of EGFP was observed and transfection efficiency was detected by Flow Cytometer. Real Time PCR and Western blotting analysis were carried out to confirm overexpression of RhoD in A375 cells. Results RhoD lentiviral vector pLV [ Exp ]-EGFP/Neo-CMV 〉 hRhoD was constructed successfully as identified by PCR and gene sequencing. Lentivirus with high- efficiency infection was produced by transfection to 293FT cells and the virus titer was （5.13 ± 2） × 10^8TU/ mL. The EGFP could be observed after the lentiviruses were transfected into A375 cells and the transfection efficiency was higher than 80%. RhoD mRNA and protein levels in RhoD overexpression cells（A375-RhoD） were significantly higher than that in cells infected by Lentiviral vector only expressing EGFP（A375-EGFP） and no infected cells （A375）. Conclusion The recombinant lentiviralvector for over-expressing RhoD was successfully constructed by Gateway technology and transfected into melanoma A375 cellsfor further experiment.