携带hFGF21基因慢病毒的制备及鉴定

发布时间：2018-06-22 20:26

本文选题：人成纤维细胞生长因子- + 慢病毒颗粒　；参考：《细胞与分子免疫学杂志》2013年12期

【摘要】：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。
[Abstract]:Objective to package human fibroblast growth factor 21 (hFGF21) lentivirus particles and determine the morphological characteristics and transduction ability of human embryonic kidney 293T (HEK293T) cells. Methods the recombinant plasmid Lv105-hFGF21 was sequenced and packaged as lentivirus particles in HEK293T cells. After identification by reverse transcription-polymerase chain reaction (RT-PCR) and electron microscope, the titer of Lv105-hFGF21 was determined by real-time quantitative PCR. Then Vero cells were infected. The expression of hFGF21 mRNA was detected by RT-PCR and the expression of hFGF21 protein was detected by immunofluorescence. Results RT-PCR results showed that recombinant lentivirus could transcribe hFGF21 mRNAs. The titer of recombinant virus was 3.68 脳 108VG / mLand the titer of concentrated solution was 1.25 脳 109VGr / mL.After the recombinant lentivirus infected Vero cells, the expression of hFGF21 mRNA was detected by RT-PCR, and the expression of hFGF21 protein was detected by immunofluorescence in Vero cells. Conclusion HFGF21 lentivirus granules were successfully packaged and expressed in target cells.
【作者单位】：中国医学科学院北京协和医学院医学生物学研究所;云南省重大传染病疫苗研发重点实验室;
【基金】：协和青年科研基金资助课题(2012D14)
【分类号】：R392.11

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