The Expression, Purification And Functional Study Of Super Positively Charged ZFN Fusion Protein In E. Coli

Human immunodeficiency virus is a human immunodeficiency virus infections Can affect the process of cellular and humoral immunity by destroying the human lymphocytes, resulting in paralysis of the immune system, there is a great threat to human health. The latest research results show that, CCR5 receptor is a T cell HIV entry co-receptor, and therefore damage the CCR5 gene in the genome or block the CCR5 gene expression has become a new way to block HIV infection of human T cells.In this paper, Ni beads and Co beads affinity chromatography purification of the fusion protein ZFN surplus positive charge, verify that the protein has cut the CCR5 gene activity, thus blocking HIV entry for the subsequent T cells and HIV gene therapy offers new ideas. The main results of this study are as follows:1.superpositively charge ZFN fusion protein expressionThe use of IPTG on the conversion of the p ET22b-(+) 36 GFP / ZFNL and p ET22b-(+) 36 YFP / ZFNR plasmid two strains were induced overnight and then incubated at different temperatures and concentrations of IPTG conditions, respectively, were the next day to collect bacteria body ultrasonication, SDS-PAGE detection of the distribution of the protein. The results determined 16 ?, 0.5m M IPTG induction of fusion protein(+)36GFP / ZFNL and(+) 36YFP/ZFNR optimal conditions expression2.superpositively charge ZFN fusion protein purification conditions of exploration1) fusion protein beads by Ni affinity chromatography purification + 36 GFP / ZFNL, found that high salt lysate conditions favorable to this fusion protein linked to the column, subsequent elution of the fusion protein can be efficiently obtained.2) + 36 YFP / ZFNR same fusion protein lysates at high salt conditions favorable to its hanging column, but subsequent elution difficulties, suggesting that the fusion protein in combination with Ni beads too strong. Then use the Co beads and beads have been used twice Ni the fusion protein was purified and found that the method can be obtained + 36 YFP / ZFNR fusion protein.3.Construction of recombinant plasmid Pet22 b / CCR5 ofPCR amplification of HCC1954 cell genomic DNA as a template, to obtain effective human CCR5 gene. CCR5 gene of interest is then ligated into plasmid vector Pet22 b, by colony PCR, double digestion and gene sequencing methods to verify the CCR5 gene without mutation.4. Super positive charge ZFN fusion protein activity detectionthe recombinant plasmid p ET22 b / CCR5 and two superpositively charge ZFN fusion protein were incubated at 37 ? 1h, 1% agarose gel electrophoresis, was found with super positive charge ZFN increasing protein molar amount of integration, restructuring plasmid p ET22 b / CCR5 amount is cut linear plasmid products also increased, indicating that ZFN protein activity enhanced functionality. At the same time, we found that when the super positive charge ZFN fusion protein molar amount increased after 7n M, the protein would cleavage recombinant plasmid p ET22b/CCR5 non-specific cleavage.In summary, we have successfully expressed super positive charge ZFN fusion protein(+) 36 GFP / ZFNL and(+) 36 YFP / ZFNR in E. coli, and by Ni affinity chromatography for the two fusion proteins were purified. On this basis, we also constructed a recombinant plasmid Pet22 b / CCR5, verify this superpositively charge ZFN fusion protein in vitro cleavage activity.This article of Super positive charge ZFN fusion protein of activity in the body forlaid a solid theoretical foundation, but also provides a new way for HIV gene therapy.