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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 encapsulate the lentiviral particles of human fibroblast growth factor 21 (hFGF21) and determine the morphological characteristics of the 293T (HEK293T) cells in packaging cells and the transduction ability of the target gene. Methods Lentiviral recombinant plasmid Lv105-hFGF21 was identified by sequencing. Lentiviral particles were packaged and concentrated in HEK293T cells. After identification by reverse transcription-polymerase chain reaction (RT-PCR) and electron microscopy, the virus titer was determined by real-time quantitative PCR. Vero cells were then infected and the expression of hFGF21 mRNA was detected by RT-PCR. The expression of hFGF21 protein was detected by immunofluorescence. Results The results of RT-PCR showed that the recombinant lentivirus could transfect hFGF21 mRNA. The results of electron microscopy showed that the typical lentivirus particle morphology was 40 ~ 70 nm in diameter. The titer of recombinant virus was 3.68 × 108VG / mL. The titer of liquid was 1.25 × 109VG / mL. The hFGF21 mRNA was detected by RT-PCR and the hFGF21 protein was detected by immunofluorescence in Vero cells after infection with recombinant lentivirus. Conclusion hFGF21 lentivirus particles were successfully packaged and expressed in target cells.