Study On The Ability Of Transporting DNA Of Histone H1 And TAT Fusion Protein

Gene Therapy is a new treatment model developed in nineties of the twentieth century. It replaced the mutation pathogenic gene, also by changing the genetic structure of disease cells, or by importing genes of enhance immunity to human body, to control the disease and achieve the purpose of treatment. And compared to traditional drug therapy, if gene therapy as a fundamental method to control disease, the vector which can be injected into the body must be the developed. At present, the gene delivery system is divided into virus carriers and non-viral vector. Virus carriers: Non-targeting, a strong immunogenicity, prone to wild-type virus. Because of the low immunogenicity and the relatively safety of Non-viral vector, it is concerned increaslly.Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vector resulted in a number of non-viral vector products, they have a flexibility of application, ease of use, low costs of production, and enhanced the safety of gene. Use the strategy of non-viral vector, oligonucleotide (ODNS) can be delivered naked (less efficient) or embedded cationic liposomes, polymers or peptide formed low buffer delivery systems, to achieve the purpose of therapy according to the target of cells. Tissues and cells internalization has been further strengthened by changing of physical or chemical method . There are many kinds of non-viral gene transfer vector, histone belongs to this family, because of their positively charge, DNA with a negative charge, they can be closely integrated through the charge, so that the structure of DNA by the extension is reduced to a relatively small volume of DNA particles.The first protein across the cell membrane to enter the cells is of human immunodeficiency virus ,HIV. Studies have shown that exogenous protein conjugated with TAT can be transported to the cells, confirmed the ability of TAT transgenic membrane. N-(47-57) of 11 amino acids of TAT protein is the domain of transduction, sequence is YGRKKRRQRRR. Search for efficient gene transfer vector, we constructed histone H1 as the skeleton of the gene delivery system, recombinant histone H1 gene, applied overlap extension PCR technology to recombinant histone gene and TAT trarsmembrane systems, build a fusion protein gene TAT-H1ct and expressed in prokaryotic cells. combinate fusion protein after purificate with pEGFP/C1 plasmid and then transfect Hela cells and fibroblast of mouse , detecte the intensity of green fluorescence to determine the ability to transfer DNA.1. Expression and purification of TG fusion gene and detection efficiency of TAT trarsmembraneApplied overlap extension PCR technique to recombinant green fluorescent protein gene and TAT gene, acquired recombinant plasmid of the fusion gene: pMD 18-T- TAT-GFP and prokaryotic expression plasmid pET-28a-TAT-GFP, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and fusion protein was detected by 12% SDS-PAGE electrophoresis, The specific TG fusion protein appeared at 31.5 kDa, conformity with the expected molecular weight, induced conditions is 1 mmol / L of IPTG final concentration, and the recombinant protein is highest expression when induced time is 6h. Fusion protein TG is purified, fusion protein is role in Hela cells. The results showed that compared with the control group, after 6 h , tumor cells with TG fusion protein can be observated high green fluorescence intensity by the laser scanning confocal fluorescence microscope, in the control group did not have green fluorescence.2 Expression of prokaryotic expression vector of TAT-H1ct fusion gene and study the ability of transfectedr DNA to the cellsApplied PCR technology to recombinant H1ct gene and TAT gene, TAT-H1ct recombinant fusion protein gene is acquired, based on the recombinant plasmid pMD18-T-TAT-H1ct of fusion gene, we constructe the recombinant expression plasmid pET-28a-TAT-H1ct successfully, the recombinant plasmid was transformed into receptor BL21 (DE3) to express, and the fusion protein was detected by 12% SDS-PAGE electrophoresis, TAT-H1ct fusion protein is appered at around 32 kDa, conformity with the expected molecular weight. Induced conditions is 1 mmol / L of IPTG final concentration, and the fusion protein is highest expression when induced time is 5h. Fusion protein TAT-H1ct is purified,. After purification, the matching conditions of fusion protein and pEGFP/C1 plasmid is optimized, detecte the ability of the fusion protein binding DNA by observate the electrophores and then transfect Hela cells and fibroblast of mouse, after 48 hours high green fluorescence is observated by laser scanning confocal fluorescence microscopy. However the control group Hela cells and mouse fibroblasts is no green fluorescence. The results show that the fusion protein can carry DNA into the cell membrane with high efficiency, have a stronger ability to transfer DNA, is expected to become a gene transfer vector.In this study, through the detection of fluorescence intensity show that TAT-H1ct has high trarsmembrane efficiency, is expected to become a gene transfer vector, and establish foundation for further study of tumor treatment.