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目的采用直接分子克隆体外连接的方法构建重组腺病毒AdHu5-040-mfsp27(cds),高效高质量扩增和纯化重组腺病毒以满足实验及临床研究的需求。方法将mfsp27克隆至穿梭质粒pSh CMV intron-eGFP两个稀有限制性内切酶之间(I-ceuI和PI-sceI)(与腺病毒骨架质粒相同),回收酶切片段后连接,用合适的感受态细胞转化后氨苄青霉素抗性挑选克隆,用2～3种合适的限制性内切酶酶切证实重组腺病毒质粒骨架和目标基因的大小和方向,必要时测序。然后线性化该重组腺病毒以释放腺病毒载体基因组的反向末端重复序列(ITRs),在HEK293细胞系转染、感染、扩增,改良氯化铯梯度离心法纯化病毒并测定病毒的滴度。结果成功构建pAdHu5-mfsp27(cds)(J1034),扩增后纯化病毒浓度为1×1013 VP/ml,MOI为1.44×1011,二者比值为69.44。重组腺病毒AdHu5-mfsp27可高效感染293包装细胞。结论用直接分子克隆的方法成功构建重组腺病毒AdHu5-mfsp27(cds)并高质量扩增,解决了用同源重组方法构建时难于挑选克隆,时间长且成功率低的问题。利用高效液相柱,生物胶联合氯化铯梯度离心技术纯化病毒,简化了操作,不需透析病毒,最大程度保存病毒活性,病毒纯度高,为研究基因的功能及实现重组腺病毒产业化提供了依据。
Objective To construct the recombinant adenovirus AdHu5-040-mfsp27 (cds) by direct molecular cloning in vitro and to efficiently and qualitatively amplify and purify recombinant adenovirus to meet the needs of experimental and clinical research. Methods mfsp27 was cloned into the shuttle plasmid pSh CMV intron-eGFP between two rare restriction enzymes (I-ceuI and PI-sceI) (the same as the adenovirus backbone plasmid). The digested fragment was recovered and ligated, Ampicillin resistant clones were selected after transformation of competent cells, and the size and orientation of the recombinant adenovirus plasmid backbone and the target gene were confirmed by 2-3 suitable restriction endonucleases and sequenced if necessary. The recombinant adenovirus was then linearized to release the inverted terminal repeats (ITRs) of the adenoviral vector genome, transfected, infected, amplified in HEK293 cell lines, and purified by cesium chloride gradient centrifugation to determine the virus titer . Results The recombinant plasmid pAdHu5-mfsp27 (cds) (J1034) was successfully constructed. The purified recombinant virus was 1 × 1013 VP / ml and the MOI was 1.44 × 1011. The ratio of the two was 69.44. The recombinant adenovirus AdHu5-mfsp27 efficiently infected 293 packaging cells. Conclusion The recombinant adenovirus AdHu5-mfsp27 (cds) was successfully constructed by direct molecular cloning, which solved the problem of difficult to select clones when constructed by homologous recombination method, long time and low success rate. The purification of the virus by high-performance liquid column and bio-gel combined with cesium chloride gradient centrifugation has simplified the operation without dialysis virus, saved the virus activity to a maximum extent and high virus purity for the study of gene function and industrialization of recombinant adenovirus The basis.