Study On The Cloning And Expression Of Fusion Gene For Pseudomonas Exotoxin A And Human Histone H3

Pseudomonas Exotoxin A is a 66,000 dalton protein toxin that kills eukaryotic cells bearing specific receptors for the toxin.lt is organized in three structural and functional domains.Domain I a is responsible for cell recognition and binding,domain II for translocation across the cell membrane,and domain III catalyses the ADP-ribosylation of elongation factor 2 (EF2),thereby inhibiting protein synthesis and leading to 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 I a and II gene segments of PEA were linded with histone H3 gene segment, then a recombinant fusion protein was constructed which serves a carrier for the transfer of DNA via receptor-mediated endocytosis. A HOObp PEA fragment which contain structural domain I a and II was amplified using cloned plasmid pCDPT2 as template by PCR. Human genome DNA was extracted then as temlate, human histone H3 gene(416bp) was amplified by PCR.The two fragment was fusioned and inserted into pMD-18T victor.Then for correct reading code frame ,the recombinant plasmid pMD-lST-PEA-Ha was cleavaged with Nco I IXho I and cloned into the expression victor pET-28c.The senses clone were subjected to restriction enzyme analysis and sequencing.The result showed that the Prokaryotic expression victor pET-28c-PEA-H3 was constructed successfully. After the expression plasmid was extracted and transformed into the expression hosts BL21(DE3) of E.coli, the transformed hosts were induced with IPTG. By SDS-PAGE and Western blot analysis of the host protein, the expression of the objective gene was detected, and it could account for 16.28% of the total host protein.Inclution body was prepared from the incubateing expression hosts induced by IPTG. After purification,the inclution body was emulsificated with Freund complete adjuvant and then inoculated rabbits 3 times with 14 day interval. By analysis of indirect ELISA, the anti-PEA-H3 antibody appeared in the rabbit serum induced by the recombinant fusion protein,and the highest antibody level was observed during 7-14days after the final inoculation.at last, rabbit anti-PEA serum with 1:1600 ELISA litre was prepared.Then,for the next functional analysis of the fusion protein and transfaction experiment,the previous work was finished.