Expression, Purification Of Histone From Hyperthermophilic Archaeon Pyrococcus Horikoshii OT3 And Preparation, Elementary Alpplication Of Its Monoclonal Antibodies

Gene therapy is the most revolutionary medical technology developed beginningfrom 1980s. In the latest decade, gene therapy as a promising tool for treatment ofhuman diseases plays a key role at the forefront of medicine. However, there are someproblems limiting the development of gene therapy, especially the gene vectortechnique. Histones, which are highly basic DNA binding proteins, probably willbecome a more effective gene vector.Pyrococcus horikoshii OT3 is a strain of hyperthermophilic archaea. In 1998, thecomplete genome sequence of this organism has been determined at the NationalInstitute of Technology and Evaluation (Tokyo, Japan). Two Open Reading FramesPHS051 and PHS046 respectively encode HPhA and HPhB which were putativearchaeal histones. Studies indicated that HPhA, a recombinant histone-like proteinfrom Pyrococcus horikoshii OT3 strain, with special structure, function and highstability has compacting activity with DNA even though under high salt or hightemperature condition. The extreme stability, conservation and DNA packagingactivity of the HPhA make it a candidate as a DNA carrier.We cultured recombinant protein HPhA engineering E.coli in fermentor toharvest about 16g/L E.coli, then lysed by sonication,identified by Tricine-SDS-PAGEelectrophoresis,the result showed that the percentage of protein expression of therecombinant protein HPhA in total soluble protein was 18%. The summarizedprocedure of purification is as followed:DNase I treatment——heat denaturation;thermal denaturation—G25 desalinization—affinity chromatograph by heparin—condensation. By this way,1L culture could be purified to attain about 2.66mgcompetent recombinant protein HPhA,the purity was about 95.35%. Electrophoreticmobility results indicated that HPhA were highly binding to DNA.BALB/c mice were intraperitoneally immunized with produced recombinantHPhA protein. We have successfully produced three strains of monoclonal antibodies(1A12,3C11,3B4) through indirect ELISA screening method and cell fusion method.Identification of subclass showed that 1A12 belonged to IgG1, 3C11 belonged toIgM and 3B4 belonged to IgG2, respectively. The ELISA titers of hybridoma culturesupernatants were 1:400,1:800 和 1:800;the ELISA titers of ascites were 1:2×105,1:9×105,1:8×106;the affinity constant were 5.36×107 L/mol,1.73×108 L/mol and2.62×108 L/mol,Relative affinity showed:3B4>3C11>1A12.Western blotting (recombinant protein PE-HPhA (PEAIa+PEAII+HPhA) asantigen, monoclonal antibody 3B4 as antibody) results showed that monoclonalantibody could specifically bind to PE-HPhA, which determined the producedmonoclonal antibody could be used to detect the recombinant protein PE-HPhA.ELISA assay detected recombinant protein HPhA in the transformed cells. So thevalidity and specificity of monoclonal antibody being used in the detection ofrecombinant protein HPhA has been confirmed. Moreover, it also proved thefeasibility of recombinant protein HPhA acting as a transfer vector.The study supplyed a detecting method for recombinant protein HPhA andPE-HPhA by preparation monoclonal antibody for the recombinant protein HPhA.