小分子蛋白慢病毒表达系统的构建

Construction of the lentiviral expression system for small proteins

目的构建趋化因子蛋白慢病毒表达系统,建立稳定表达GFP的卵巢癌细胞系,为研究趋化因子蛋白的功能及作用机制打下基础。方法趋化因子CCL18、IL-8、CXCL1基因克隆质粒经PCR扩增、酶切,与慢病毒载体PWPI连接,构建的重组慢病毒质粒按比例与包膜质粒PCMV-dR8.74及包装质粒PMD2.G混合,LipofectinTM2000共转染293T细胞系包装病毒颗粒,病毒液感染卵巢癌SKOV3细胞后,采用流式细胞仪分选出GFP表达的细胞作为转染成功的细胞。实时荧光定量PCR验证转染后目的基因的表达,Westernblot验证目的蛋白的表达。结果重组慢病毒基因能高效地转导入靶细胞,转导效率达95%以上,并稳定表达;实时荧光定量PCR检测结果显示:细胞和组织中转染目的基因组与对照组比较,均见目的基因的表达显著增高(P<0.05);Westernblot验证转染目的基因组小分子蛋白成功表达。结论本实验成功构建了小分子蛋白慢病毒表达系统,为进一步研究小分子蛋白在人体的功能及作用机制建立了实验基础。

Objective For further study the function and mechanism of chemotactic protein, to establish a lentiviral expression system and to construct a human ovarian cancer cell line that can express green fluorescent protein （GFP） stably. Method- s Chemotactic factor CCLI8 ,IL-8 and CXCL1 gene plasmids were amplified by PCR, restricted by endonuclease digestion and connected with lentiviral vector PWPI. The recorabinant lentiviral vector was then mixed with the envelope plasmid PCMV- dR8.74 and packaging plasmids PMD2. G in proportion. LipofectinTm2000 293T cells were co-transfection with packaging viral particles. The viruses harvested from the superuatant were used to transfect ovarian cancer SKOV3 cells. The cells that strongly expressed GFP were selected by FACS,which served as the successfully transfected cells. The target genes were measured by Real-time fluorescent quantitative PCR. Western blot was adopted to confirm the expression levels of the target proteins. Results The efficiency of infection of the cells that express target gene was more than 95%. Real-time fluorescent quantitative PCR tests showed that the expression level of target gene improved significantly comparing to the control cells and tissues （ P 〈 0. 05 ）. The expression of chemotactic protein in the transfected cells was validated by Western blot. Conclusion A lentiviral expression system for small protein is successfully constructed, which is an experimental basis for further study the function and mechanism of small-molecule protein.