穿膜肽-LacI HPM载体的构建及其DNA结合活性测定

摘要

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, LacI HP(headpiece), LacI HPM(headpiece mu-tant) 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 di-alyzed 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 concentrat-ed, 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.%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、LacI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，在缓冲液中氧化获得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（+）-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-LacI 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：构建了穿膜肽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。