| 摘要: |
| ［摘要］ 目的 构建靶向RelA（p65）基因的短发夹RNA（short hairpin RNA,shRNA）慢病毒载体，并对其功能进行验证。方法 设计3对针对RelA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，经菌落聚合酶链式反应（PCR）及测序鉴定，将重组正确的质粒进行慢病毒包装和滴度测定，通过Western blot实验筛选出对HepG2细胞株中RelA基因干扰效果最好的慢病毒，并通过CCK-8检测Lenti-shRelA对细胞增殖活性的影响。结果 测序结果显示重组慢病毒载体与设计参考序列一致，提示重组慢病毒载体构建成功。重组慢病毒Y4056、Y21318、Y21319、Y21320的滴度分别为3.13×108 TU/ml、2.97×108 TU/ml、2.51×108 TU/ml、3.40×108 TU/ml。用慢病毒Lenti-shRelA（Y21318、Y21319、Y21320、Y4056）感染HepG2细胞，Western blot实验结果显示Y21320对HepG2细胞株中RelA基因的干扰效果最好。CCK-8实验结果显示RelA的敲降可显著抑制细胞增殖活力。结论 该研究成功构建了靶向RelA基因shRNA慢病毒载体，其能有效下调HepG2细胞RelA的表达并抑制细胞增殖，为进一步研究RelA在肝癌发生、发展中的机制奠定了基础。 |
| 关键词: RelA基因 短发夹RNA 慢病毒 肝癌 |
| DOI：10.3969/j.issn.1674-3806.2023.09.07 |
| 分类号:R 735.7 |
| 基金项目:北京市自然科学基金项目（编号：7192084）；首都卫生发展科研专项项目（编号：2020-2-1152）；北京市属医学科研院所公益发展改革试点项目（编号：京医研2021-10） |
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| Construction and functional verification of shRNA lentiviral vector targeting to RelA(p65) gene |
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HUO Yun-fei, GAO Ming-hui, DOU Shuang-shuang, et al.
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Beijing Institute of Hepatology, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China
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| Abstract: |
| ［Abstract］ Objective To construct a short hairpin ribonucleic acid(shRNA) lentiviral vector targeting to RelA(p65) gene and to verify its function. Methods Three pairs of RNA interference sequences targeting to RelA gene were designed, and their corresponding shRNA sequences were synthesized. After annealing of shRNA to form double-stranded oligo sequence, the recombinant plasmid was constructed by gene recombination technique. The correct recombinant plasmid was used for lentivirus packaging and titer determination after polymerase chain reaction(PCR) and sequencing identification of the colony were performed. The lentivirus with the best interference effect on RelA gene in HepG2 cell line was screened by Western blot, and the effect of Lenti-shRelA on cell proliferation viability was detected by cell count kit-8(CCK-8). Results The results of sequencing analysis showed that the recombinant lentivirus vector was consistent with the designed reference sequence, suggesting that the recombinant lentivirus vector was successfully constructed. The titers of the recombinant lentiviruses of Y4056, Y21318, Y21319 and Y21320 were 3.13×108 TU/ml, 2.97×108 TU/ml, 2.51×108 TU/ml and 3.40×108 TU/ml, respectively. After lentiviruses Lenti-shRelA(Y21318, Y21319, Y21320, Y4056) were infected with HepG2 cells, the experimental results of Western blot showed that Y21320 had the best interference effect on RelA gene in HepG2 cell line. The experimental results of CCK-8 showed that RelA knockdown significantly inhibited cell proliferation viability. Conclusion In this study, the shRNA lentiviral vector targeting to RelA gene is successfully constructed, which can effectively down-regulate the expression of RelA in HepG2 cells and inhibit the cell proliferation, laying a foundation for further research on the mechanism of RelA in the occurrence and development of liver cancer. |
| Key words: RelA gene Short hairpin ribonucleic acid(shRNA) Lentivirus Liver cancer |
