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Title:: 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2; 董鹏1; 张静1; 焦杨1; 郭永红3△; 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

Author(s):: WANG Junhong1; WEN Jiao2; DONG Peng1; ZHANG Jing1; JIAO Yang1; GUO Yonghong3; 1.Department of Endocrinology , the Second Affiliated Hospital,Medical School of Xi’an Jiaotong University, Xi’an 710004,China；2.Department of Oncology, the Second Affiliated Hospital,Medical School of Xi’an Jiaotong University, Xi’an 710004；3.Department of Infectious Disease, the Second Affiliated Hospital,Medical School of Xi’an Jiaotong University, Xi’an 710004

Keywords:: Exendin-4; Double stranded adeno-associated virus; Single stranded adeno-associated virus; Diabetes mellitus; Adeno-associated virus

摘要:: 比较双链腺相关病毒（Double-stranded adeno-associated virus, dsAAV)及单链AAV（Single-stranded adeno-associated virus, ssAAV)介导Exendin-4分泌表达效率。应用基因工程方法构建dsAAV/pSSHG/Exendin-4，与重组ssAAV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dsAAV pSSHG/Exendin-4滴度为2.5×1011pfu，较重组ssAA滴度高(2.5×109 pfu)；dsAAV/ pSSHG/ GFP转染NIH3T3细胞表达绿色荧光强；ELISA法检测感染重组dsAAV的NIH3T3细胞上清中Exendin-4的浓度为181.1±8.75pmol/ml，较重组ssAAV分泌浓度（133.81±8.09pmol/ml）升高（〖WTBX〗P〖WTBZ〗<0.05）；重组dsAAV及ssAAV转染NIH3T3细胞Exendin-4表达均为阳性。重组dsAAV可分泌表达Exendin-4并较ssAAV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。

Abstract:: To construct a recombinant double-strands adeno-associated virus（dsAAV）expressing Exendin-4，compared transfecting 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 recombinant virus was by homologous recombinationin dsAAV （2.5×1011pfu）than by ssAAV（2.5×109pfu）. There was higher fluorescence 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 expression system in dsAAV has relatively high transfecting ability. It should play a significant and fundamental role for further researches about diabetes gene therapy.