摘要
目的：构建带有人对氧磷酶1（hPONI）基因的重组腺相关病毒（rAAV）载体，并检测其转染大鼠骨髓内皮祖细胞（EPCs）后hPON1的表达。方法：提取流产胎儿肝脏的总RNA，RT-PCR获得cDNA，设计引物通过PCR获得PON!基因片段，将其与pGEM—TEasy载体连接，将PON1基因片段切下，插入到质粒pAAV—IRES—GFP的多克隆位点，获得质粒pAAV—PON1-IRES—GFP，经酶切和测序鉴定。包装的病毒rAAV—PON1用斑点杂交检测滴度，并用其转染EPCs检测hPON1在mRNA水平的表达。结果：pAAV—PON1-IRES-GFP酶切和测序结果完全正确。斑点杂交检测病毒的滴度为1．7×10^10g／mL，RT—PCR结果显示病毒转染EPCs后存在hPON1的表达。结论：成功构建了pAAV—PON1-IRES—GFP质粒，包装出较高滴度的rAAV—PON1，并且转染EPCs后可以检测到hPON1表达。
Objective：To construct a recombinant adeno-associated virus （rAAV） vector containing human paraoxonase-1 （hPONI） gene, and to detect the expression of hPON1 after transfecting EPCs derived from rat bone marrow. Methods： Total RNAs were extracted from human fetal liver and cDNA were obtained by RT-PCR. PON1 gene was obtained by a further PCR using specifically designed primers, bound to pGEM-T Easy, and then inserted into the muhiple clone sites of an adeno-associated viral plasmid pAAV-IRES-GFP to construct an adenoassociated viral plasmid pAAV-PONI-IRES-GFP （ pAAV-PONI ）. The plasmid pAAV-PON1 was identified by digesting with restriction enzyme and sequence analysis. The recombinant AAV-PONI-IRES-GFP （ rAAV-PON1 ） was packaged through co-transfecting HEK293 cells with plasmid pAAV-PON1, pAAV-RC and pHelper, and purified with the method single-step column purification （SSCP）. The titer was detected by dot-blot. Results： The plasmid pAAV-PONI-IRES-GFP proved to be identical with restriction enzyme digestion and sequence analysis. The titer of rAAV was 1.77 × 10^10 v. g/mL . RT-PCR analysis demonstrated that PON1 mRNA was expressed in PONl-transduced EPCs. Conclusion： Recombinant adeno-associated virus containing PON1 was successfully constructed, which could transfect EPCs and express the PONlgene in transfected EPCs.
出处
《广州医学院学报》
2010年第5期57-62,共6页
Academic Journal of Guangzhou Medical College
关键词
腺相关病毒
动脉粥样硬化
对氧磷酶1
内皮祖细胞
recombinant adeno-associated virus
atherosclerosis
paraoxonase-1
endothelial progenitorcells
