携带hFGF21基因慢病毒的制备及鉴定

Preparation and characterization of the recombinant hFGF21-containing lentiviral

目的包装人成纤维细胞生长因子21(hFGF21)慢病毒颗粒、确定包装细胞人胚肾293T(HEK293T)细胞的形态特征以及对目的基因的转导能力。方法对慢病毒重组质粒Lv105-hFGF21进行测序鉴定,在HEK293T细胞中包装为慢病毒颗粒并浓缩,经反转录PCR(RT-PCR)和电镜检测鉴定后,利用实时定量PCR测定病毒滴度。然后感染Vero细胞,经RT-PCR检测hFGF21 mRNA的表达、免疫荧光检测hFGF21蛋白的表达。结果 RT-PCR结果表明重组慢病毒能转录hFGF21 mRNA,电镜检测结果可以看到典型的慢病毒颗粒形态特征,其直径在40～70 nm;重组病毒原液的滴度是3.68×108VG/mL、浓缩液的滴度是1.25×109VG/mL;重组慢病毒感染Vero细胞后经RT-PCR检测到细胞中有hFGF21mRNA表达,免疫荧光测到Vero细胞中有hFGF21蛋白表达。结论成功包装了hFGF21慢病毒颗粒并在靶细胞中获得表达。

Objective To package the lentiviral particles carrying human fibroblast growth factor21 （hFGF21） and identifythe morphological characteristics and transduction capability for the target gene of human embryo kidney 293T （HEK293T）cells. Methods After sequencing identification, the lentiviral recombinant plasmid Lvl05-hFGF21 was packaged andconcentrated in HEK293T cells, and then identified by RT-PCR and electron microscopy. The titer of recombinant lentiviruswas determined by real-time quantitative PCR. The expression of hFGF21 at both mRNA and protein levels was detected byRT-PCR and immunofluorescence, respectively in lentivirus-infected Vero cells. Results RT-PCR demonstrated that hFGF21mRNA could be transcribed in Lvl05-hFGF21 infected HEK293T cells. The typical lentiviral particle was seen under theelectron microscope. The diameter of virus particles was 40-70 nm. The title of the virus was substantially elevated from 3.68xlO8 VG/mL to 1.25 × 109 VG/mL after concentration. The expression of hFGF21 was detected in Lv-hFGF21 infected Verocells by means of both RT-PCR and immunofluorescence. Conclusion The hFGF21 lentiviral particles have been packagedsuccessfully and hFGF21 can be expressed in the infected Vero cells.