Research On Construction, Expression And Characteristic Of A New Gene Delivery Vector RPEA-HPhA

In recent years, many gene delivery vectors have been extensively used, but they almost have inherent limitations and disadvantages, and deliver their genes passively, rely on uptake into vesicular compartments by endocytosis. Cationic polypeptides, such as poly-arginine and poly-lysine, have been reported to bind DNA, form complexes with DNA, and introduce themselves into cells. Their efficiency for practical applications to gene therapy, however, remains untested. The results are similar to past reports showing that gene transfer via receptor-mediated by endocytosis or mediated by DEAE-dextran is markedly enhanced with endosomotrophic agent, such as chloroquine. However, efficient gene transfer such as is necessary for genetic analysis has not yet been successfully achieved. A novel method still needs to be developed so that gene can be transferred in vitro and in vivo, then genetic analysis can be carried out in higher eukaryotes. Receptor-mediated DNA transfer may be one such method.Pseudomonas exotoxin A (PEA) is a single chain protein, which consist of three domains. domainâ… a is composed of residues 1-252 ,which is responsible for cell recognition. domainâ…¡is composed of residues 253-364, which is involved in translocation of the toxin across membranes. domainâ…  b, residues 365-404 and its function is still unclear. domainâ…¢is composed of residues 405-613, it catalyzes the ADP-ribosylation of elongation factor-2, which arrests protein synthesis and results in cell death. The deletion of domanâ…¢doesn't have an influence on the acceptor binding and the translocation of PEA when it goes throught the cell membrane and into the cytosol. The receptor binding domain of PEA is a highly specific ligand to the LDL/a2-macroglobulin receptor of all mammalian cells. The membrane translocation domain of PEA can increase the translocation efficiency of DNA. We utilized these domains of receptor binding and membrane translocation in the development of a general DNA delivery vehicle that has no cell-type specificity. Then, we need a DNA binding region that has no DNA sequence and topological specificity for this DNA delivery vehicle.Pyrococcus horikoshii OT3 strain is a hyperthermophilic archaeon. The genome sequening of this organism has been completed at the National Institute of Technology and Evaluation. Two open reading frames PHS051 (HPhA) and PHS046 (HPhB) were putative archaeal histones. According to the gene sequence, the analysis of bioinformation and the pre-experimental research, it was concluded that the HPhA gene can encode a kind of histone-like protein. The result of gel electrophoresis mobility shift assays demonstrated that the purified HPhA has high affinity for DNA. The HPhA can increase linear plasmid electrophoresis mobilities in proper HPhA/DNA ratio, and the DNA was no limited to DNA sequence and topological specificity. So we can utilize HPhA-DNA binding characterization as a DNA binding protein domain for the construction of a gene delivery vehicle.Here, we develop a novel DNA delivery system that has the high target cell specificity, no cell-type specificity, and no restriction in DNA size and structure, and could deliver initiatively foreign gene. We, therefore, fused the receptor binding and membrane translocation domains of PEA with the DNA binding region of the N-terminal 70 amino acid residues of recombinant HPhA.Initially, a recombinant plasmid pET26b-PEAIa+II-HPhA was obtained successfully and a protein was expressed in E.coli BL21(DE3)-codon plus .It consisted of domainâ… a ,domainâ…¡of PEA and HPhA. Through optimizing the fermentation condition from E coli stain, suitable culture medium, IPTG concentration, culture time, we obtain the cells of weight reached 17g/L.The cells was collected by centrifugation and lysed by sonication. The protein was precipitated by 35% ammonium sulfate and digested by DNaseâ… , then purified by Blue Chelating Sepharose and Heparin Fast Flow chromatogphy. The purity of rPEA-HPhA was about 90%. Gel electrophoresis mobility shift assays demonstrated rPEA-HPhA is same as HPhA, which can increase linear plasmid electrophoresis mobilities. The speciality of the recombinant rPEA-HPhA affinity for DNA was explored.The fusion protein rPEA-HPhA had no cytotoxicity to BHK and Hela cells, it could transfer HPhA into the Hela cells by immunochemistry determination. We used pEGFP-rPEA-HPhA complex act on CHO cells. through the fluorescence microscope, we could observe the transfection of fusion protein rPEA-HPhA had higher efficiency than cationic liposome in CHO cell lines . All showed rPEA-HPhA protein can mediate transfection in vitro. The research of fusion protein rPEA-HPhA was very important to gene delivery vector.According to the type of carried gene, the target direction of the rPEA-HPhA protein can be changed, in order to reduce the cytotoxic of gene therapeutic. It is keys to the study of gene transfection in vivo for the future.Taken together our results suggested that the rPEA-HPhA has the characteristic of an ideal vector: efficient, nontoxic, targeting, and bypass the endocytic pathway to minimize DNA degradation. So we believed the study of rPEA-HPhA as the vector delivering gene voluntarily may have the potential to be used in gene therapy or cancer therapy, but more experiments need to be conducted in the future in order to maximize the transfection of this fusion protein in vivo.