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  

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

作　　者：胡榕[1] 吴可贵 许昌声 

机构地区：[1]福建医科大学附属协和医院心内科,福州350001 [2]福建福建省高血压研究所

出　　处：《中国临床药理学与治疗学》2005年第4期391-396,共6页Chinese Journal of Clinical Pharmacology and Therapeutics

摘　　要：目的:构建表达大鼠核因子κB(NF- κB ,p6 5 )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(spontaneouslyhypertensiverat,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 (p6 5 )cDNA片段(16 5 3bp) ,用TA克隆技术插入pMD18 T载体,构建pMD18 T/16 5 3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(p6 5 ) 3’端2 6 9bp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(+ ) /2 6 9质粒含有NF κB(p6 5 )的2 6 9bp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/2 6 9。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/2 6 9。线性化的重组腺病毒质粒转染2 93细胞后产生的重组腺病毒颗粒用于VSMCs的感染。WesternBlot测定感染前后VSMCs中NF- κB(p6 5 )表达的变化情况。结果:构建的pMD18 T/16 5 3重组质粒经过序列测定,证实其包含大鼠NF κB(p6 5 )基因的16 5 3bp片断;pcDNA3.1(+ ) /2 6 9、pENTR4 /CMV/2 6 9、pAd/CMV/2 6 9等重组质粒经内切酶消化或PCR等方法鉴定无误;AIM: To construct the recombinant adenovirus 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 was used 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 subsequently 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 vector, pcDNA3.1(+), under the CMV promoter. The intact antisense RNA expression frame from pcDNA3.1 (+)/269 was inserted into entry vector pENTR4 to form medium recombinant pENTR4/CMV/269 plasmid, followed by homologous recombination technique, the expression frame was integrated into adenovirus. The linearized recombinant adenovirus plasmid, pAd/CMV/269, infected the monolayer 293 cells, the adenovirus packaging 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 analysis was documented as expected. The transition- vectors contained 269 bp of reverse sequence were identified by restrictive endonuclease and PCR analysis. Recombinant adenovirus that expressed NF-κB (p65) antisense 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 infected VSMCs was markedly reduced compare with that in the normal cells. CONCLUSION: Recombinant adenovirus that expressed NF-κB (p65) antisense RNA is constructed successfully and the virus possesses the biological feature of down-regulated expression of NF-κB (p65) in the infect

关 键 词：核因子-KB 反义RNA 腺病毒载体 表达调 控 蛋白免疫印迹 大鼠 

分 类 号：R966[医药卫生—药理学]