重组双链及单链腺相关病毒介导Exendin-4表达效率的比较  

The Contrast Tranfecting Efficiency of a Double Stranded Recombinant Adeno- associated Virus Expressing Exendin- 4 Compared with Single Stranded AAV

作　　者：王俊红[1] 问姣 董鹏[1] 张静[1] 焦杨[1] 郭永红[3] 

机构地区：[1]西安交通大学医学院第二医院内分泌科,西安710004 [2]西安交通大学医学院第二医院肿瘤科,西安710004 [3]西安交通大学医学院第二医院感染科,西安710004

出　　处：《生物医学工程研究》2015年第2期122-127,共6页Journal Of Biomedical Engineering Research

基　　金：国家自然科学基金青年基金资助项目(81100300);西安交通大学自由探索与自主创新类项目(Xjj20100018)

摘　　要：比较双链腺相关病毒(Double-stranded adeno-associated virus,ds AAV)及单链AAV(Singlestranded adeno-associated virus,ss AAV)介导Exendin-4分泌表达效率。应用基因工程方法构建ds AAV/p SSHG/Exendin-4,与重组ss AAV比较转染NIH3T3细胞效率及转染后细胞上清Exendin-4浓度,免疫组化检测Exendin-4表达。经限制性内切酶及测序证实载体构建成功,测定重组ds AAV p SSHG/Exendin-4滴度为2.5×1011pfu,较重组ss AA滴度高(2.5×109pfu);ds AAV/p SSHG/GFP转染NIH3T3细胞表达绿色荧光强;ELISA法检测感染重组ds AAV的NIH3T3细胞上清中Exendin-4的浓度为181.1±8.75pmol/ml,较重组ss AAV分泌浓度(133.81±8.09pmol/ml)升高(P<0.05);重组ds AAV及ss AAV转染NIH3T3细胞Exendin-4表达均为阳性。重组ds AAV可分泌表达Exendin-4并较ss AAV具有更高效转染能力,为研究Exendin-4功能及应用于临床打下基础。To construct a recombinant double -strands adeno -associated virus（dsAAV）expressing Exendin -4,compared trans-fecting efficiency in cells with a single stranded AAV（ssAAV）.The recombinant dsAAV vector pSSHG/Exendin -4 was reconstructed which Exendin -4 by gene engineering method.The recombinant virus was packaged by human embryo kidney 293 cells and the virus titer was measured .The expression GFPs of dsAAV and ssAAV in NIH3T3 cell were detected by fluorescence microscope.Exendin -4 level in the superment of NIH3T3 transferred by dsAAV /pSSHG/Exendin -4or ssAAV /pSSHG/Exendin -4 were determined by ELISA.The expression Exendin -4 of dsAAV and ssAAV in NIH3T3 cell were detected by immunohistochemical.Recombinant dsAAV vector was successfμl constructed and confirmed by restriction enzymes and DNA sequencing.The more higher titer of recombi-nant virus was by homologous recombinationin dsAAV （2.5 ×10 11 pfu）than by ssAAV（2.5 ×109 pfu）.There was higher fluorescence＆amp;nbsp;intensity in transfected NIH3T3 cells by dsAAV.The Exendin -4 level was higer in supernatant of dsAAV than in ssAAV（181.1 ± 8.75 pmol/mlvs133.81±8.09 pmol/ml）.The Exendin -4 expression was positive both in dsAAV and ssAAV.The Exendin -4 ex-pression system in dsAAV has relatively high transfecting ability.It should play a significant and fundamental role for further researches about diabetes gene therapy.

关 键 词：EXENDIN-4 双链AAV 单链AAV 糖尿病 腺相关病毒 

分 类 号：R587.1[医药卫生—内分泌]