体外靶向TP53BP2基因shRNA慢病毒载体的构建及功能鉴定*

doi:10.3969/j.issn.1672-5069.2023.02.004

实用肝脏病杂志 ›› 2023, Vol. 26 ›› Issue (2): 164-168.doi: 10.3969/j.issn.1672-5069.2023.02.004

体外靶向TP53BP2基因shRNA慢病毒载体的构建及功能鉴定^*

霍云飞, 寇卜心, 柴梦音, 豆双双, 高明慧, 石英, 刘晓霓

100069 北京市首都医科大学附属北京佑安医院北京肝病研究所

收稿日期:2022-10-14 出版日期:2023-03-10 发布日期:2023-03-21
通讯作者: 石英,E-mail:yingshi@ccmu.edu.cn
作者简介:霍云飞,男,23岁,硕士研究生。E-mail:yunfeihuo@mail.ccmu.edu.cn
共同第一作者:寇卜心,女,32岁,大学本科,检验技师。E-mail:koubuxin@sina.com
基金资助:
^*北京市自然科学基金资助项目(编号:7192084);首都卫生发展科研专项(编号:2020-2-1152);北京市属医学研究所公益发展改革试点项目(就医研2021-10)

Construction and functional verification of a shRNA lentiviral vector targeting to TP53BP2 gene in HepG2 cells in vitro

Huo Yunfei, Kou Buxin, Chai Mengyin, et al.

Beijing Institute of Hepatology, You’an Hospital, Capital Medical University, Beijing 100069, China

Received:2022-10-14 Online:2023-03-10 Published:2023-03-21

摘要/Abstract

摘要： 目的本研究旨在构建靶向肿瘤蛋白p53结合蛋白2(TP53BP2)基因的短发夹RNA(shRNA)慢病毒载体,以抑制肝癌细胞TP53BP2的表达。方法设计了2对针对TP53BP2基因的RNA干扰序列,并合成相应的shRNA序列。shRNA退火形成双链oligo序列后,应用基因重组技术构建重组质粒,经菌落PCR和测序鉴定,将重组正确的质粒进行慢病毒包装和滴度测定,并采用Western Blot、qRT-PCR和激光共聚焦技术观察慢病毒Lenti-shTP53BP2对HepG2细胞TP53BP2基因的干扰效果。结果测序比对结果显示,各重组慢病毒载体与设计参考序列一致,提示各重组慢病毒体构建成功;重组慢病毒载体经慢病毒包装后,显示pHS-ASR-LW429、pHS-ASR-LW512和pHS-ASR-LW513的滴度分别为9.7×10⁸ TU/mL、6.1×10⁸ TU/mL和6.4×10⁸ TU/mL;用慢病毒Lenti-shTP53BP2(pHS-ASR-LW512和pHS-ASR-LW513)感染HepG2细胞后,与对照慢病毒(pHS-ASR-LW429)比,经Western Blot、qRT-PCR和激光共聚焦结果显示两个Lenti-shTP53BP2均能显著下调HepG2细胞TP53BP2基因水平和蛋白表达量。结论本研究成功构建了靶向TP53BP2基因shRNA慢病毒载体,其能有效下调HepG2细胞TP53BP2的表达,为进一步研究TP53BP2在肝癌发生发展过程中的机制研究奠定了基础。

关键词: HepG2细胞, 肿瘤蛋白p53结合蛋白2, 短发夹RNA, 慢病毒, 体外

Abstract: Objective The present paper aimed to inhibit the expression of tumor suppressor p53-binding protein 2 (TP53BP2) in liver cancer by short hairpin RNAs(shRNAs) with lentiviral vector. Methods Two pairs of RNA interference sequences targeting to TP53BP52 gene were designed, and their corresponding shRNA sequences were synthesized. After annealing of shRNA to form double-stranded oligo sequences, the recombinant plasmid was constructed by gene recombination technique. The correct recombinant plasmid was used after PCR and sequencing identification of the colony for lentivirus packaging and titer determination. The interference effect of lentivirus lenti-shTP53BP2 on TP53BP2 gene in HepG2 cells was observed by Western Blot, qRT-PCR and laser confocal technique. Results The sequencing alignment results showed that each recombinant lentiviral vector was consistent with the designed reference sequence, indicating that each recombinant lentiviral vector was successfully constructed; the titers of pHS-ASR-LW429, pHS-ASR-LW512 and pHS-ASR-LW513 were 9.7×10⁸ TU/mL, 6.1×10⁸ TU/mL and 6.4×10⁸ TU/mL, respectively; the HepG2 cells were infected with lentivirus lenti-shTP53BP2 (pHS-ASR-LW512 and pHS-ASR-LW513), and the results of Western blot, qRT-PCR and laser confocal technique showed that the two lenti-shTP53BP2 significantly down-regulated the TP53BP2 RNA level and its protein expression in HepG2 cells as compared with the control lentivirus (PHS-ASR-LW429). Conclusion In this study, we successfully construct the shRNA lentiviral vector targeting to TP53BP2 gene with the capacity of effectively down-regulation of TP53BP2 expression in HepG2 cells, which might lay a foundation for further research on the mechanism of TP53BP2 in the hepatocarcinogenesis.

Key words: HepG2 cells, Tumor suppressor p53-binding protein 2, Short hairpin RNA, Lentivirus, In vitro

霍云飞, 寇卜心, 柴梦音, 豆双双, 高明慧, 石英, 刘晓霓. 体外靶向TP53BP2基因shRNA慢病毒载体的构建及功能鉴定^*[J]. 实用肝脏病杂志, 2023, 26(2): 164-168.

Huo Yunfei, Kou Buxin, Chai Mengyin, et al.. Construction and functional verification of a shRNA lentiviral vector targeting to TP53BP2 gene in HepG2 cells in vitro[J]. Journal of Practical Hepatology, 2023, 26(2): 164-168.

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体外靶向TP53BP2基因shRNA慢病毒载体的构建及功能鉴定^*

Construction and functional verification of a shRNA lentiviral vector targeting to TP53BP2 gene in HepG2 cells in vitro

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