穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

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，构建新型基因转导载体。方法：PCR扩增LacI、Lacl基因前头肽序列、前头肽序列突变体、TAT序列的编码基因，构建前头肽序列突变体和TAT的原核表达载体，可溶性表达TAT-LacI HPM融合蛋白并纯化，在缇冲液中氧化获得TAT-LacI HPM二聚体并浓缩，PCR检测二聚体融合蛋白与质粒的体外结合能力。结果：获得了pET-28a（-4-）-LacI HPM及pET-28a（＋）-TAT-LacI HPM表达质粒，表达纯化并获得二聚化融合蛋白，体外结合实验确定TAT-Lac／HPM二聚体融合蛋白与检测质粒DNA具有特异的高亲和力结合活性。结论：构建了穿膜呔TAT-LacI HPM，为进一步研究其作为新型DNA转运载体的可行性奠定了基础。

Construct of Cell-Penetrating Peptides-LacI HPM Vectors and Detection of its DNA-Binding Activity

Objective： To construct the novel DNA delivery vectors by using cell-penetrating peptides with the ability of high efficient cellular penetration and LacI to specifically bind to its recognizing DNA sequence with high affinity. Methods： The DNA sequence encoding the LacI, LaeI HP（headpiece）, LacI HPM（headpiece mutant） and TAT were firstly amplified by PCR respectively, then the expression plasmids of TAT-LacI HPM were cloned into pET-28a（＋） vectors. Fusion proteins expressed in E.coli were purified by Ni-NTA beads, and were dialyzed against buffer to form the TAT-LacI HPM dimers, then the dimers were concentrated by PEG-8000. DNA-binding activities were examined. Results： The expression plasmids of pET-28a（＋）-LacI HPM and pET-28a （＋）-TAT-LacI HPM were obtained, after expression and purification, the TAT-LacI HPM dimers were concentrated, and the results showed that TAT-LacI HPM dimers have high affinity with its recognizing DNA sequences. Conclusion： The TAT-LacI HPM fusion proteins may have the potential to serve as a novel DNA delivery vector.