携带前强啡肽基因的Ad5 F35重组腺病毒制备及鉴定

目的 用Ad5F35载体制备携带前强啡肽基因的重组腺病毒Ad5F35-PDP并对其进行鉴定.方法 利用NheI和Agel分别对合成的目的 基因prodynorphin聚合酶链反应(PCR)产物和pDC316-LacZ-a载体质粒进行双酶切.将PDP目的 基因片段和线性化的pDC316连接,克隆构建pDC316-PDP.pDC316-PDP与腺病毒骨架pBHG-fiber5/35共转染293细胞制备Ad5F35-PDP重组腺病毒,收获并纯化病毒,检测重组病毒滴度,运用PCR进行鉴定.结果 编码PDP的基因序列经测序鉴定证实与Gene Bank中的一致,pDC316-PDP构建成功.pDC316-PDP与腺病毒骨架共转染293细胞后见明显的毒斑,说明两者在293细胞中同源重组并包装成功.经PCR证实Ad5F35-PDP重组腺病毒构建完成.病毒滴度为1×1012v.P./ml.结论 本实验成功制备了含PDP全序列的高滴度、高纯度的Ad5F35-PDP重组腺病毒,为下一步进行该重组病毒生物安全性能和生物学功能研究及今后应用Ad5F35-PDP重组腺病毒对吗啡成瘾患者进行基因治疗奠定了基础.

Construction and identification of recombinant adenovirus Ad5F35 containing prodynorphin gene

Objective To construct and identify recombinant adenovirus expressing prodynorphin gene (PDP) in vector Ad5 F35.Methods Restriction fragment of PDP from pUC57-PDP was inserted in the vector of pDC316.The insertion and the direction of the insert were confirmed by polymerase chain reaction (PCR) and restriction enzyme digestion.The constructed pDC316-PDP was co-transfected with adenovirus backbone pBHG-fiberS/35 into 293 cells to establish the recombinant adenovims Ad5F35-PDP, which was confirmed by PCR.Results The gene sequence coding PDP was identical with that of GenBank by DNA sequencing, and the construction of pDC316-PDP was completed.The significant virus plaques was observed in the 293 cells co-transfected with pDC316-PDP and pBHG-fiber5/35, indicating the successful homologue recombination and virus packaging of the interest fragment in 293 cells.The successful construction of the recombinant adenovirus Ad5F35-PDP with PCR was confirmed, and the titer of Ad5F35-PDP was approximately 1 × 1012 v.p./ml.Conclusion The high-titer,purified recombinant adenovirus of Ad5F35-PDP which contains the full-length coding region of PDP has been established, which could be used in the gene therapy for morphine addiction, and to test the bio-safety and biological function of Ad5F35-PDP in the future.