大鼠核因子-κB 反义 RNA 腺病毒载体的构建及其在血管平滑肌细胞中的表达

目的: 构建表达大鼠核因子 κB(NF-κB, p65)反义 RNA 的重组腺病毒, 并测定受染血管平滑肌细胞(VSMCs) 中 NF-κB 基因的表达变化。方法: 从自发性高血压大鼠(spontaneously hypertensive rat, SHR)的VSMCs 中提取总 RNA, 经 RT-PCR 方法获得含全部编码序列的NF-κB (p65) cDNA 片段(1 653 bp), 用TA 克隆技术插入 pMD18-T 载体, 构建 pMD18-T/1653 重组质粒, 并经酶切和 DNA 测序鉴定。以该质粒为模板, 经 PCR 获得 NF-κB(p65) 3' 端 269 bp 片段, 逆向插入 pcDNA3.1(+) 真核表达载体并置于CMV 启动子下。由此构建的 pcDNA3.1(+)/269 质粒含有NF-κB(p65) 的269 bp反义RNA 完整表达框。随后将该表达框克隆到入门载体 pENTR4, 构建重组质粒 pENTR4/CMV/269。使用同源重组方式在体外将该表达框重组到腺病毒载体 pAd/PL-DEST, 最终得到重组腺病毒质粒 pAd/CMV/269。线性化的重组腺病毒质粒转染 293细胞后产生的重组腺病毒颗粒用于VSMCs 的感染。Western Blot 测定感染前后VSMCs 中NF-κB(p65) 表达的变化情况。结果: 构建的pMD18-T/1653 重组质粒经过序列测定, 证实其包含大鼠NF-κB(p65) 基因的 1653 bp 片断;pcDNA3.1(+) /269、pENTR4/CMV/269、pAd/CMV/269 等重组质粒经内切酶消化或 PCR等方法鉴定无误;线性化的 pAd/CMV/269 转染 293 细胞后, 可产生完整 、具有感染性的重组腺病毒颗粒, 病毒在感染上清中的滴度 为 9.23 ×10⁹ pfu°ml^-1 (plaque form units permilliliter);Western Blot 检测证实, 感染重组腺病毒的VSMCs中的NF-κB(p65) 蛋白水平明显减少。结论: 构建了可表达大鼠 NF-κB (p65) 反义 RNA 的重组腺病毒 Ad/CMV/269, 该病毒在受染的 VSMCs 内具有下调 NF-κB (p65) 基因表达的生物学功能, 为后续的基因治疗研究奠定基础。

Construction of recombinant adenovirus vector encoding the antisense RNA of rat NF-κB and its expression in vascular smooth muscle cells

AIM: To construct the recombinant aden-ovirus contained antisence RNA of rat nuclear factor κB (NF-κB, p65) gene and investigate the change of the gene expression in the infected vascular smooth muscle cells (VSMCs). METHODS: Total RNA was extracted from VSMCs of spontaneously hypertensive rats (SHR) and it wasused for amplifing the 1653 bp fragment of NF-κB (p65) gene by RT-PCR.The amplified product was inserted into the pMD18-T vector and identified subse-quently by enzyme digestion analysis and sequencing. The downstream 269 bp of NF-κB (p65) gene was amplified from the recombinant pMD18-T/1653 plasmid and cloned in reverse orientation into the eukaryotic expression vec-tor, pcDNA3.1(+), under the CMV promoter.The in-tact antisense RNA expression frame from pcDNA3. 1 (+) /269 was inserted into entry vector pENTR4 to form medium recombinant pENTR4/CMV/269 plasmid, fol-lowed by homologous recombination technique, the ex-pression frame was integrated into adenovirus.The lin-earized recombinant adenovirus plasmid, pAd/CMV/269, infected the monolayer 293 cells, the adenovirus packag-ing cell line. Western blot was employed to determine the changes of NF-κB (p65) gene expression level in the virus-infected VSMCs.RESULTS: A 1653 bp fragment of NF-κB (p65) gene was amplified from VSMCs, and its sequence analysiswas documented as expected.The tran-sition-vectors contained 269 bp of reverse sequence were identified by restrictive endonuclease and PCR analysis. Recombinant adenovirus that expressed NF-κB (p65) an-tisense RNA was constructed correctly and the titer of virus was generally up to 9.23 ×10⁹ plaque form units per milliliter (pfu°ml^-1). In vitro, western blot results showed that the NF-κB (p65) expression level in the in-fected VSMCs was markedly reduced compare with that in the normal cells.CONCLUSION: Recombinant aden-ovirus that expressed NF-κB (p65) antisense RNA is con-structed successfully and the virus possesses the biological feature of down-regulated expression of NF-κB (p65) in the infected VSMCs.The production of the recombinant adenovirus encoding NF-κB (p65) antisense RNA pro-vides a foundation for investigation of its activity and fur-ther gene therapy.