【作者】 张梅；

【导师】 何金生；

【作者基本信息】 安徽医科大学 ， 免疫学， 2004， 硕士

【摘要】 目的 腺病毒AdEasy系统是一种利用原核细胞完成重组穿梭质粒与腺病毒骨架质粒之间的同源重组，快速构建重组腺病毒的好方法。我们将其中的某些步骤进行了改进，以获得一种更加高效、简便、快速构建重组腺病毒的方法。 方法 我们将腺病毒骨架载体pAdEasy-1通过电穿孔法转化E．coli BJ5183感受态细胞，经抗性筛选、酶切鉴定获得含pAdEasy-1的E．coli BJ5183／p菌种，并鉴定其稳定性。随后用CaCl₂法制备E．coli BJ5183／p感受态细胞。并转化含GFP的腺病毒穿梭质粒，利用E．coli BJ5183细胞内同源重组机制获得重组腺病毒质粒，经酶切鉴定并确定重组的位置。与常规的共转化方法相比较，了解这种方法的重组效率。将鉴定正确的重组腺病毒质粒进一步转化E．coli DH10B感受态细胞，获得高拷贝的重组质粒。以Pac Ⅰ酶切，获得重组腺病毒DNA分子，脂质体法转染293细胞，产生基因组结构均一的重组腺病毒。感染原代血吸虫细胞进行腺病毒嗜性的初步研究。 结果 利用改进的方法，获得了具有高度稳定性可重复使用的E．coli BJ5183／p菌种，经CaCl₂法制备为感受态细胞后，可方便地用于转化重组穿梭质粒，并获得重组腺病毒质粒。与常规方法相比较，该法的重组效率有一定的提高。重组腺病毒质粒DNA分子转染293细胞，可观察到293细胞出现肿胀，圆缩等典型的细胞病安徽医科大学硕士学位论文变（C PE），荧光显微镜观察细胞内有大量绿色荧光。进一步感染原代血吸虫细胞，表明腺病毒对其有一定的嗜性。结论改进后的方法构建重组腺病毒更加简便，重组效率较原方法提高，可在国内普通的分子生物学和病毒学实验室内完成构建工作。更多还原

【Abstract】 Objective: Adenovirus AdEasy system is a good method for rapid constructing recombinant adenovirus by employing homologous recombination between recombinant shuttle plasmid carrying interesting gene and adenovirus backbone plasmid in prokaryocyte. In order to construct recombinant adenovirus more easily, we try to make the protocol improved and obtain a more efficient, convenient and rapid method.Method: The replication-defective adenovirus type 5 backbone plasmid pAdEasy-1 was transformed into competent E.coli BJ5183 by electroporation. The resultant E.coli BJ5183/p strain was selected with ampicillin and screened by restriction endonuclease digestion, and its stability was assessed by restriction endonuclease digestion. Then the E.coli BJ5183 / p competent cells were prepared by CaCl2 technique and the recombinant shuttle plasmid carrying GFP (pAdTrack-CMV) was transformed into them. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. The recombination efficiency between recombinant shuttle plasmid carrying interesting gene and adenovirus backbone plasmid was compared to the routine method of co-transformation. The obtained recombinant adenovirus was further transformed into competent E.coli DH 10B in order to obtain high-copy recombinant plasmid. The confirmed recombinant adenovirus DNA was linearlized withPac I and transfected into 293 cells line to produce the recombinant adenovirus (rAdv). We also investigated the infection capability of the rAdv to primary schistosoma culture cells.Result: By using the improved method, we obtained the E.coli BJ5183 / p strain with high stability and it can be used repeatedly. The competent E.coli BJ5183 / p prepared by CaCl2 technique was easily transfomed by recombinant shuttle plasmid, and recombinant adenovirus plasmid can be produced by homologous recombination in E.coli BJ5183 / p. The recombination efficiency has been improved compared to the routine method. After the recombinant adenovirus DNA was transfected into 293 cells line, and the CPE of 293 cells and the green fluorescence were observed. The rAdv can infect some kind of primary schistosoma culture cells.Conclusion: This improved method for constructing recombinant adenovirus is more convenient and more efficient than the routine method. It can be applied well in a poorly equipped laboratory in our country.更多还原