RHBDF2基因过表达慢病毒载体的构建和稳定表达RHBDF2细胞系的建立

doi:10.13481/j.1671-587x.20200303

摘要/Abstract

摘要： 目的：构建RHBDF2基因过表达慢病毒载体，建立稳定表达RHBDF2的EA.hy926细胞，为RHBDF2基因过表达慢病毒载体的构建和稳定表达RHBDF2细胞的建立提供依据。方法：根据NCBI提供的RHBDF2基因序列，设计并合成引物，PCR扩增RHBDF2基因后通过Gateway克隆技术将目的基因克隆至入门载体，再亚克隆至慢病毒载体pLV[Exp]-EGFP，构建重组慢病毒质粒pLV[Exp]-EGFP-RHBDF2；将慢病毒表达载体质粒pLV[Exp]-EGFP和重组慢病毒质粒pLV[Exp]-EGFP-RHBDF2分别与慢病毒辅助包装质粒共转染HEK293T细胞，包装慢病毒并测定慢病毒滴度；将感染pLV[Exp]-EGFP-control的EA.hy926细胞作为对照组，感染pLV[Exp]-EGFP-RHBDF2的EA.hy926细胞作为实验组，利用嘌呤霉素筛选出稳定表达RHBDF2的EA.hy926细胞；荧光定量PCR （qPCR）和Western blotting法检测对照组和实验组EA.hy926细胞中RHBDF2 mRNA和蛋白表达水平。结果：酶切电泳和测序检测，实验组过表达慢病毒载体的基因序列与设计合成的序列完全一致；对照组包装的慢病毒滴度为1×10⁸ TU·mL^-1，实验组慢病毒的滴度为3×10⁸ TU·mL^-1；荧光显微镜下慢病毒成功感染EA.hy926细胞，感染率达95%以上；qPCR检测，实验组EA.hy926细胞中RHBDF2 mRNA表达水平较对照组明显升高（P<0.01）；Western blotting法检测，实验组EA.hy926细胞中RHBDF2蛋白表达水平较对照组明显升高（P<0.05）。结论：成功构建RHBDF2过表达慢病毒载体，利用pLV[Exp]-EGFP-RHBDF2慢病毒建立了稳定上调RHBDF2表达的EA.hy926细胞系。

关键词: RHBDF2, 慢病毒载体, HEK293T细胞, EA.hy926细胞

Abstract: Objective: To construct the RHBDF2 gene over-expression lentivirus vector and to establish the EA.hy926 cells stably expressing RHBDF2, and to provide the evidence for the construction of RHBDF2 gene over-expression lentivirus vector and the establishment of RHBDF2 cells stably expressing RHBDF2. Methods: According to the sequence of RHBDF2 gene provided by NCBI,and the primers were designed and synthesized; the RHBDF2 gene was amplified by PCR method, and the target gene was cloned into the entry vector by Gateway cloning technology, and then subcloned into the lentivirus vector pLV[Exp]-EGFP to construct the recombinant lentivirus plasmid pLV[Exp]-EGFP-RHBDF2; the lentivirus expression vector plasmid pLV[Exp]-EGFP and the recombinant lentivirus plasmid pLV[Exp]-EGFP-RHBDF2 were co-transfected into the HEK293T cells with the virus-assisted packaging plasmids to package the lentivirus and the titer of the lentivirus was detected. The EA.hy926 cells infected with pLV[Exp] - EGFP-control were used as control group and the EA.hy926 cells infected with pLV[Exp] - EGFP-RHBDF2 were used as experiment group. The EA.hy926 cells stably expressing RHBDF2 were screened by puromycin. The fluorescent quantitative PCR (qPCR) and Western blotting methods were used to detect the expression levels of RHBDF2 mRNA and protein in the EA.hy926 cells in control group and experiment group. Results: The enzyme digestion electrophoresis and sequencing results showed that the gene sequence of the EA.hy926 cells over-expression lentivirus vector in experiment group was completely consistent with the designed and synthesized sequence. The lentivirus titer in control group was 1×10⁸ TU·mL^-1, and the lentivirus titer in experiment group was 3×10⁸ TU·mL^-1. The EA.hy926 cells were successfully infected with the lentivirus under fluorescence microscope and the infection efficiency was above 95%.The qPCR detection results showed that the expression level of RHBDF2 mRNA in the EA.hy926 cells in experiment group was higher than that in control group (P<0.01).The Western blotting results showed that the expression level of RHBDF2 protein in the EA.hy926 cells in experiment group was higher than that in control group (P<0.05). Conclusion: The lentivirus vector over-expressing RHBDF2 is successfully constructed, and the EA.hy926 cell line stably up-regulating the expression of RHBDF2 is established by using pLV[Exp]-EGFP-RHBDF2 lentivirus.

Key words: RHBDF2, lentivirus vector, HEK293T cells, EA.hy926 cells

中图分类号:

R543.5

陈少凤, 李胜男, 邓福, 朱佩仪, 李友. RHBDF2基因过表达慢病毒载体的构建和稳定表达RHBDF2细胞系的建立[J]. 吉林大学学报(医学版), 2020, 46(03): 444-450.

CHEN Shaofeng, LI Shengnan, DENG Fu, ZHU Peiyi, LI You. Construction of RHBDF2 gene over-expression lentivirus vector and establishment of RHBDF2 cell line stably expressing RHBDF2[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 444-450.

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