靶向型辅助腺病毒的构建及其功能研究

Construction of retargeting helper adenovirus and its functions

目的构建一种新型辅助腺病毒,并用于靶向型第三代腺病毒载体的制备,提高其对造血细胞的感染效率。方法采用重叠PCR的方法合成含有不完全包装信号序列A1-A4和loxP序列的DNA片段SynES,替换穿梭质粒pShuttle中原有的包装信号序列,得到穿梭质粒pShuttle-SynES;将所得穿梭质粒与骨架质粒Ad5/F11p在大肠杆菌BJ5183中同源重组,获得重组腺病毒质粒载体pAd5/F11p-HV,将其转染293细胞包装重组腺病毒Ad5/F11p-HV。参照第三代腺病毒包装策略,利用Ad5/F11p-HV包装获得携带绿色荧光蛋白(GFP)基因的第三代腺病毒HD-Ad5/F11p-GFP;将其以不同的感染强度感染人白血病细胞系K562、U937、Jurkat和人脐带血CD34+细胞后,采用荧光显微镜和流式细胞术检测GFP的表达。结果采用DAN片段SynES替换穿梭质粒pShuttle上的包装信号,获得新的穿梭质粒pShuttle-SynES;进一步构建获得重组腺病毒质粒pAd5/F11p-HV,并制备了辅助腺病毒Ad5/F11p-HV。采用该辅助腺病毒包装pC4HSU-GFP,获得了第三代腺病毒HD-Ad5/F11p-GFP;CsCl密度梯度离心分离HD-Ad5/F11p-GFP和Ad5/F11p-HV,获得了高质量的HD-Ad5/F11p-GFP。与对照病毒HD-H14-GFP相比,HD-Ad5/F11p-GFP可明显提高对人白血病细胞U937、Jurkat和人脐带来源CD34+细胞的感染效率。结论设计并构建了一种靶向性辅助腺病毒,并以此为基础成功制备了对造血细胞高效感染的第三代重组腺病毒。

Objective To construct a novel helper adenovirus, which could package targeting helper-dependent adenovirus （HD-Ad）, and to improve its gene transfer efficiency in hematopoietic cells. Methods The packaging signal sequences of pShuttle were replaced by the DNA fragment SynES containing the sequences of A1-A4 and loxP, which was synthesized by overlapping polymerase chain reaction （PCR）. The new pShuttle plasmid pShuttle-SynES was homologous recombined with Ad5/F1 l p in B J5183 strains to generate recombinant adenoviral vector pAd5/F1 l p-HV. Then, Ad5/Fllp-HV was prepared in HEK293 cells after transfected by liposomes. The HD-Ads, HD-Ad5/Fllp-GFP, carrying green fluorescent protein gene was rescued according to the instructions of helper-dependent adenovirus system from Microbix. Human leukemia cell lines, K562, U937, Jurkat and CD34＋ cells derived from human cord blood were infected by HD-Ad5/F1 lp-GFP at serial multiplicity of infection （MOI）, and the gene transduction efficiency was detected by fluorescence microscope and flow cytometry. Results We successfully constructed DNA fragment SynES and cloned into the corresponding sites in pShttule. The plasmid pAd5/F1 l p-HV was generated in B J5183 strains, and then the adenovirus Ad5/F1 l p-HV was successfully prepared in 293 cells. Ad5/Fllp-HV packaged the helper-dependent adenoviral vector pC4HSU-GFP and then generated HD-Ad5/Fllp-GFP. HD-Ad5/F 1 l p-GFP was successfully separated from Ad5/F 11 p-HV by CsC1 density gradient centrifugation. Compared to the control of HD-H14-GFP, the gene transfer ability of HD-AdS/F1 lp-GFP to hematopoietic cells was improved obviously both in leukemia cell lines and CD34＋ cells derived from human cord blood. Conclusion We successfully prepare a helper-dependent adenovirus, which is able to infect hematopoietic cells efficiently, based on a targeting helper adenovirus.