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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p LV/helper-SL3,pLV/helper-SL4及p LV/helper-SL5混合采用脂质体法制备DNA-Lipofectamine 2 000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RhoD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/NeoCMV>h RhoD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/m L。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RhoD mRNA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 containing human RhoD gene and construct a lentivirus vector. The recombinant plasmid was transfected into human melanoma A375 cells to overexpress RhoD protein, which lays the foundation for further studies on the role of RhoD in melanoma. Methods Gateway technique was used to construct the RhoD lentiviral vector carrying enhanced green fluorescent protein EGFP. After being identified by PCR and gene sequencing, the recombinant plasmid was co-transfected with the helper plasmids p LV / helper-SL3, pLV / helper-SL4 and p LV / helper- The DNA-Lipofectamine 2000 complex was prepared by plastid method and co-transfected into 293FT cells for lentivirus packaging to generate the corresponding lentivirus particles. The virus titer was determined by quantitative PCR. The packaged lentiviral vector was transfected into human melanoma A375 cells. Fluorescence microscopy was used to observe the expression of fluorescence. The transfection efficiency was detected by flow cytometry. The experiment was divided into A375 (untreated control), A375-EGFP (Control group) and A375-RhoD (virus group with RhoD gene) were used to verify the effect of RhoD in A375-RhoD group by real-time fluorescence quantitative PCR (QPCR) and western blotting expression. Results By PCR, gene sequencing confirmed that lentiviral expression vector pLV [Exp] -EGFP / NeoCMV> h RhoD was successfully constructed. 293FT cells were cotransfected with the helper plasmids to produce a highly infectious lentivirus. The virus titer was (5.13 ± 2) × 10 ~ 8TU / m L. The transfection efficiency of A375-RhoD group was significantly higher than that of A375-EGFP group and A375 group. The expression of RhoD mRNA and protein in A375-RhoD group was significantly higher than that in A375-EGFP group and A375 group. Conclusion The RhoD lentiviral vector was successfully constructed and packaged with highly infectious lentiviral particles and successfully transfected into human melanoma A375 cells, providing experimental basis for further study on the role of RhoD in melanoma.