表达绿色荧光蛋白报告基因重组禽腺联病毒载体系统的构建与鉴定

Construction and Identification of Recombinant Avian Adeno-associated Virus Expressing GFP Reporter Gene

为了开展重组禽腺联病毒(Recombinant avian adeno-associated virus,rAAAV)介导的基因转移研究,用限制性内切酶消化含AAAV全基因组的重组质粒pCR-AAAV,去除AAAV Rep和Cap蛋白编码序列,将PCR扩增的绿色荧光蛋白(Green fluorescent protein,GFP)报告基因插入AAAV末端反向重复序列(Inverted terminal re-peats,ITR)之间,获得表达GFP基因的AAAV转移载体pAITR-GFP;以含AAAV全基因组的质粒为模板,用PCR分别扩增AAAV Rep、Cap和Rep-Cap蛋白基因,将Rep和Cap基因分别插入真核细胞双表达载体pVITRO2-mcs的两个多克隆位点,将Rep-Cap蛋白基因插入真核细胞表达载体pcDNA3,获得AAAV辅助质粒pVITRO2-ARC和pcDNA-ARC;将pAITR-GFP、pVITRO2-ARC或pcDNA-ARC与腺病毒辅助质粒pHelper组成三质粒转染系统,用磷酸钙沉淀法共转染AAV-293细胞,获得了表达GFP的rAAAV。经SDS-聚丙烯酰胺凝胶电泳分离后,纯化病毒出现分子量正确的VP1、VP2、VP3结构蛋白;经PCR检测证明,重组病毒中含有GFP报告基因;用重组病毒分别感染鸡胚成纤维细胞(CEF)和鸡胚肝CEL细胞,可以观察到GFP报告基因的表达,表达时间持续两周以上。这些试验结果表明,成功建立了辅助病毒非依赖性rAAAV体外包装体系,为禽源细胞的基因转移研究和禽重组活载体疫苗的研制打下了基础。

To generate recombinant avian adeno-associated virus （rAAAV） for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast （CEF）cells and CEL cell line were transduced with the recombinant virus , the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.