重组双链及单链腺相关病毒介导Exendin －4表达效率的比较

摘要

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 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.%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2．5×1011 pfu，较重组 ssAA 滴度高（2．5×109 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±8．75pmol ／ml，较重组 ssAAV 分泌浓度（133．81±8．09pmol ／ml）升高（P ＜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。