生物医学工程研究
生物醫學工程研究
생물의학공정연구
JOURNAL OF BIOMEDICAL ENGINEERING RESEARCH
2015年
2期
122-127
,共6页
王俊红%问姣%董鹏%张静%焦杨%郭永红
王俊紅%問姣%董鵬%張靜%焦楊%郭永紅
왕준홍%문교%동붕%장정%초양%곽영홍
Exendin-4%双链AAV%单链AAV%糖尿病%腺相关病毒
Exendin-4%雙鏈AAV%單鏈AAV%糖尿病%腺相關病毒
Exendin-4%쌍련AAV%단련AAV%당뇨병%선상관병독
Exendin-4%Double stranded adeno -associated virus%Single stranded adeno-associated virus%Diabetes mellitus%Adeno -associated virus
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。
比較雙鏈腺相關病毒(Double -stranded adeno -associated virus,dsAAV)及單鏈 AAV(Single -stranded adeno -associated virus,ssAAV)介導 Exendin -4分泌錶達效率。應用基因工程方法構建 dsAAV /pSSHG/Exendin -4,與重組 ssAAV 比較轉染 NIH3T3細胞效率及轉染後細胞上清 Exendin -4濃度,免疫組化檢測 Exendin -4錶達。經限製性內切酶及測序證實載體構建成功,測定重組 dsAAV pSSHG/Exendin -4滴度為2.5×1011 pfu,較重組 ssAA 滴度高(2.5×109 pfu);dsAAV /pSSHG/GFP 轉染 NIH3T3細胞錶達綠色熒光彊;ELISA 法檢測感染重組 dsAAV 的 NIH3T3細胞上清中 Exendin -4的濃度為181.1±8.75pmol /ml,較重組 ssAAV 分泌濃度(133.81±8.09pmol /ml)升高(P <0.05);重組 dsAAV 及 ssAAV 轉染 NIH3T3細胞Exendin -4錶達均為暘性。重組 dsAAV 可分泌錶達 Exendin -4併較 ssAAV 具有更高效轉染能力,為研究Exendin -4功能及應用于臨床打下基礎。
비교쌍련선상관병독(Double -stranded adeno -associated virus,dsAAV)급단련 AAV(Single -stranded adeno -associated virus,ssAAV)개도 Exendin -4분비표체효솔。응용기인공정방법구건 dsAAV /pSSHG/Exendin -4,여중조 ssAAV 비교전염 NIH3T3세포효솔급전염후세포상청 Exendin -4농도,면역조화검측 Exendin -4표체。경한제성내절매급측서증실재체구건성공,측정중조 dsAAV pSSHG/Exendin -4적도위2.5×1011 pfu,교중조 ssAA 적도고(2.5×109 pfu);dsAAV /pSSHG/GFP 전염 NIH3T3세포표체록색형광강;ELISA 법검측감염중조 dsAAV 적 NIH3T3세포상청중 Exendin -4적농도위181.1±8.75pmol /ml,교중조 ssAAV 분비농도(133.81±8.09pmol /ml)승고(P <0.05);중조 dsAAV 급 ssAAV 전염 NIH3T3세포Exendin -4표체균위양성。중조 dsAAV 가분비표체 Exendin -4병교 ssAAV 구유경고효전염능력,위연구Exendin -4공능급응용우림상타하기출。
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.