| 摘要: |
| 目的 制备靶向锌指和BTB结构域蛋白20（ZBTB20）基因的干扰腺病毒。方法 根据ZBTB20基因序列设计人鼠通用的干扰序列，定向克隆至腺病毒穿梭载体pShuttle-U6-GFP的BamH Ⅰ和Hind Ⅲ酶切位点，获得重组穿梭载体pShuttle-shZBTB20。将Pme Ⅰ酶切线性化的重组穿梭载体pShuttle-shZBTB20导入含有腺病毒载体pAdEasy-1的BJ5183细菌，使其同源重组产生重组腺病毒载体pAd-shZBTB20。用Pac Ⅰ酶切线性化该重组腺病毒载体，转染293A细胞包装腺病毒颗粒，用半数组织培养物感染量（TCID50）法鉴定病毒滴度，并在人肝癌Huh7细胞和小鼠肝脏组织中验证该病毒的干扰效率。结果 成功构建了人鼠通用的ZBTB20基因干扰腺病毒载体，获得高活性的ZBTB20干扰腺病毒Ad-shZBTB20，滴度为3.2×1010 IU/mL。该干扰腺病毒感染人肝癌细胞株96 h可使ZBTB20 mRNA降低至对照的20%；小鼠尾静脉注射重组病毒（1×1010 VG/只）7 d后可下调肝脏内源性ZBTB20蛋白的表达，并使其靶基因甲胎蛋白mRNA表达水平升高约203倍。结论 所制备的ZBTB20干扰腺病毒能有效抑制人和小鼠ZBTB20的表达及其生物学效应。 |
| 关键词: ZBTB20 RNA干扰 重组腺病毒 甲胎蛋白 |
| DOI：10.16781/j.CN31-2187/R.20220891 |
| 投稿时间：2022-11-22修订日期：2023-01-07 |
| 基金项目:国家自然科学基金重点项目（81130084，31730042）. |
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| Preparation and functional characterization of ZBTB20-interfering adenovirus |
| LIN Jie1△,YUAN Jing-hao2△,MA Xian-hua1,WAN Xiao-qing1,ZHANG Wei-ping1* |
(1. Department of Pathophysiology, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. School of Stomatology, Wenzhou Medical University, Wenzhou 325027, Zhejiang, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To prepare an interfering adenovirus targeting zinc finger and BTB domain containing 20 (ZBTB20) gene. Methods The human and mouse interference primers were designed based on the ZBTB20 gene sequence, and cloned into BamH Ⅰ and Hind Ⅲ restriction endonuclease sites of the adenovirus shuttle vector pShuttle-U6-GFP. The shuttle plasmid pShuttle-shZBTB20 was linearized by Pme Ⅰ and then transformed into pAdEasy-1/BJ5183 bacterial cells for generating homologous recombinant adenovirus plasmid pAd-shZBTB20. The recombinant adenovirus plasmid was linearized with Pac Ⅰ and transfected into HEK-293 packaging cells. The titer of virus was measured by tissue culture infectious dose 50 (TCID50) assay. The interference efficiency of endogenous ZBTB20 gene was detected in Huh7 cells and liver tissue in the mice. Results The adenovirus vector targeting human and mouse ZBTB20 gene was successfully constructed, and the highly active ZBTB20 interfering adenovirus Ad-shZBTB20 was obtained, with the titer of 3.2×1010 IU/mL. The expression of endogenous ZBTB20 mRNA was significantly reduced to 20% of the control in human hepatoma cell line Huh7 infected with the interfering adenovirus for 96 h. After 7 d of tail vein injection (1×1010 VG/mouse), ZBTB20 protein was dramatically down-regulated in the liver, and its downstream target gene α-fetoprotein mRNA level was increased by 203 times. Conclusion The prepared ZBTB20-interfering adenovirus can effectively inhibit the expression of human and mouse ZBTB20 and its biological effects. |
| Key words: ZBTB20 RNA interference recombinant adenovirus alpha fetoprotein |
