目的：借助穿膜肽TAT高效跨膜的特性和LacI前头肽突变体（LacI HPM）高亲和力结合DNA的特性，建立一种安全高效、无基因插入片段大小限制的基因转导系统。方法：在TAT-LacI HPM片段C端和N端分别添加GST标签，构建pET-28a（+）-TAT-LacI HPM-GST和pGEX-GST-TAT-LacI HPM重组表达载体，可溶性表达TAT-LacI HPM-GST及GST-TAT-LacI HPM融合蛋白并纯化，获得TAT-LacI HPM二聚体，免疫荧光检测TAT-LacI HPM融合蛋白穿过HeLa细胞膜的情况，观察EGFP的表达，用免疫印迹检测TAT-LacI HPM融合蛋白介导质粒DNA进入细胞的能力。结果：表达、纯化并获得二聚体融合蛋白，体内实验表明其具有跨膜能力，能介导带有LacI结合序列的DNA质粒进入细胞，并在转染细胞里检测到了目的蛋白。结论：初步证实TAT-LacI HPM融合蛋白作为一种新型通用性非病毒DNA转运载体的可行性，为评价这种新型DNA疫苗载体在提高免疫效果方面的可行性奠定了前期实验基础。%Objective: To establish a novel DNA delivery system which is safe, high efficient and no limitation of inserting DNA fragments, by using cell-penetrating peptides TAT with the ability of high efficient cellular pene-tration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods: GST-tag was inserted into either C- or N-terminal of TAT-LacI HPM(LacI headpiece mutant) fragment to obtain the expres-sion plasmids. TAT-LacI HPM fusion proteins expressed in E.coli were purified by glutathione Sepharose 4B beads, and were dialyzed against buffer to form the TAT-LacI HPM dimers. Whether the transduced dimeric TAT-LacI HPM fusion proteins could penetrate in cells was tested by immunofluorescence, and the ability of TAT-LacI HPM dimers to deliver DNA into the cells were examined by observing the expression of the reporter EGFP gene and by Western blotting. Results: The expression plasmids of pET-28a(+)-TAT-LacI HPM-GST and pGEX-GST-TAT-LacI HPM were obtained, after expression and purification, the dimers were concentrated, the transduced dimeric TAT-LacI HPM fusion proteins could penetrate in cells and mediate EGFP transfection and ex-pression in human cells. Conclusions: Our data suggest that the TAT-LacI HPM fusion proteins may have the po-tential to serve as a novel safe, efficient DNA delivery system which may improve the application of DNA vac-cines in the future.
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