Expression And Purificatin Of Fusion Gene Of Pseudomonas Exotoxin A And Human Histone H3

Pseudomonas exotoxin A (PEA) is a single chain toxin,PEA is made up of three domains. The pHysical boundaries indicated that domain Ia is composed of residues l-252;domain II, residues 253-364; domainIb, residues 365-404; and domain III, residues 405-613. It has been shown that domain Ia is responsible for cell recognition, domain II is to be involved in translocation of the toxin across membranes,and domain HI catalyzes the ADP-ribosylation of elongation factor2, Which arrests protein synthesis and results in cell death. Arginine rich human histone H3 is a kind of basic nucleosome protein.In the medium of pHysiological condition,by electrostatic interaction,histone H3 which arginine impart to it a positive charge binds to DNA which pHospHate groups impart to it a negative charge.In this paper,In order to establish a new technology of transfection, the functional domains la and II gene segments of PEA were lined with histone H3 gene segment, then a recombinant fusion protein was constructed which serves as carrier for transfer of DNA via receptor-mediated endocytosis.In this study,the recombinant plasmid pMD-18T-PEA-H3 was cleavaged with NcoI ,XhoI and inserted into the expression vector pET-28c and subsequently subjected to restriction endonuclease analysis and sequencing, the result indicated that the prokaryotic expression vector pET-28c-PEA-H3 was constructed successfully.After the expression plasmid was extracted and transformed into expression hosts BL21(DE3) of E.coli,the transformed hosts were induced by IPTG,bySDS-PAGE and ELISA analysis of host protein.the expression of theobjective gene was detected ,and it could account for 16.28% of the total host protein .inclusion body was prepared from the incubating expression hosts induced by IPTG. After purification and absolv in 8mol/L urea .And then injected rabbits 3 times with 14 days interval.By analysis of indirect ELISA, the anti-PEA-H3 antibody appear in the rabbit serum induced by the recombinant fusion protein,the highest antibody level was observed during 7-14days after the final inoculation .Incubate the genetic engineering bacteria in LB medium(Kana) and induce the inclusion body with IPTG ,then sonicated the bacterial cells and then subjected to denaturation in 8mol/L urea.Then purified inclusion body with His trap?metal affinity chromatograpHy.In order to obtain higher purity the expressed protein.Protein was further purified by CNBr-activated SepHarose 4 Fast Flow with nonspecial affinity chromatograpHy .At last,PEA-H3protein eventually collect 30%,it was prepared for next experiments functional analysis of the fusion proteinand genetic transfaction.