重复水高压注射介导hHGF表达载体在小鼠体内的持续高效表达  

Sustained and high expression of human hepatocyte growth factor in mice following repeated hydrodynamic injections of naked plasmid

作　　者：朱传龙[1] 李毓雯[2] 高人焘[1] 李宜[1] 聂青和[3] 

机构地区：[1]安徽医科大学附属安徽省立医院感染病科,合肥市230001 [2]安徽医科大学附属安徽省立医院儿科,合肥市230001 [3]第四军医大学唐都医院传染病科,全军感染病诊疗中心

出　　处：《实用肝脏病杂志》2009年第2期81-83,123,共4页Journal of Practical Hepatology

基　　金：安徽省教育厅自然科学研究项目(KJ2008B067);安徽省科技厅科技攻关项目(07010302195)

摘　　要：目的构建hHGF表达载体并通过水高压注射法研究hHGF在小鼠体内的持续表达。方法从慢性乙型肝炎患者肝组织提取总RNA,RT-PCR扩增出hHGF基因的cDNA片段,将目的片断克隆至pMD18-T载体,酶切和测序鉴定,然后将目的片断酶切回收后插入pcDNA3.1质粒,构建真核表达载体pcDNA-hHGF,通过重复水高压注射法将pcDNA-hHGF导入小鼠肝脏,并在首次水高压注射后第0、3、6、9、12、15、18和21天分别收集小鼠血清和肝组织,采用双抗体夹心酶联免疫吸附法检测hHGF在小鼠体内的表达情况。结果扩增出了长约2187bp的基因片段,并将其克隆至pMD18-T载体,酶切和测序鉴定无误;将pMD-hHGF中hHGF基因亚克隆至pcDNA3.0载体,酶切鉴定无误;首次水高压注射后不同时间检测外周血和肝脏均有hHGF的高水平表达。结论成功构建了hHGF真核表达载体pCMV-hHGF,重复水高压注射法可将该基因导入小鼠肝脏并获得持续高效的表达。Objective To construct human hepatocyte growth factor expression vector （pCMV-hHGF） and characterize the expression of pMD-hHGF in vivo by repeated hydrodynamic injections in mice. Methods Total RNA was extracted from liver tissue of a patient with chronic hepaitis B,cDNA was obtained by reverse transcription,hHGF cDNA was amplified and cloned into pMD18-T vector and the sequences were identified by retriction endonucleases and sequencing assay, hHGF gene was dissected from pMD-hHGF and recloned into pcDNA3.0, pCMV-hHGF was analyzed by restriction endonucleases to ensure the orientation. After the plasmid was transfected into mouse livers by repeated hydrodynamic injections,we collected plasma and livers of mouse at day 0,3,6,9,12,15,18 and 21 after the first hydrodynamic injection,and then detected the expression of hHGF by ELISA. Rusults A 2187bp gene fragment was obtained and coloned into pMD18-T vector,and the sequence was correct, hHGF gene was subcloned into pcDNA3.0 vector,and then restriction endonucleases assays showed the correct orientation. The level of hHGF expression could be detected by en- zyme linked immunosorbent assay after the first hydrodynamic injection. Conclusion hHGF expression vector （pCMV- hHGF） has been successfully constructed and repeated hydrodynamic injections can promote sustained and high expression of hHGF in mice.

关 键 词：促肝细胞生长因子 肝脏 水高压注射 基因表达 小鼠 

分 类 号：R575[医药卫生—消化系统]