Preparating The Antibody Of Heme Oxygenase-1 And Screening Its Epitops

Heme oxygenase-1, as one of three heme oxygenase, is the only one can be induced in vivo, and it is the rate-limiting enzyme which can degrade heme to CO, biliverdin and ferri ion. It is highly-expressed when induced by many factors, e.g. heme, heat, heavy metals, hormone, UV, oxygen deficiency, NO and so on. The induced HO-1 can exert anti-inflammatory effect and can regulate apoptosis. Many researches indicated that HO-1 is correlated with a great of diseases, e.g. tumor, enteritis, pyemia, oxygen-induced convulsion, degenerative disease of neuron, brain injured by ischemia, atherosclerosis, asthma, hypertension and so on. In vivo, HO-1 provide help in some disadvantage condition, e.g. endotoxic shock, (in tela) hyperoxia, acute pleurisy, organ transplantation and ischemia- reperfusion injury. Evidences in lots of disease models showed that HO-1 and its products(bilirubin/ biliverdin) can regulate its protect effect by their characteristics of anti-inflammatory, anti-apoptosis, anti-proliferation and antioxygen. The study on structure and function of heme oxygenase-1 (HO-1) are very important for the pathologic mechanism and therapeutics of the related diseases. Neutralization antibodies can neutralize activity of antigens, thus could be used in study of function of antigens. If epitopes of these antibodies are screened out, then the epitopes can be used in immunotherapy and pathology investigation of correlated diseases.We express HO-1 prokaryoticly and obtain highly-purified bioactive HO-1, then prepare neutralization antibody and finally get neutralization epitopes peptides by screening. This project will make a foundation for the clinical diagnosis, therapeutics and prognosis of related diseases. In this project, we determine the optimal expression condition in E. coli and purification condition of human HO-1 (hHO-1) and recombinant rat HO-1 (ΔrHO-1), and we prepare the neutralization antibody by using the purified protein. After that, epitopes ofΔrHO-1 were screened out by Phage Display, and then we prepare antibody of epitopes peptides and test the neutralize activity.First, pMW172/hHO-1 and pMW172/ΔrHO-1 were identified by digesting and sequencing. Then the correct plasmids were transformed into E. coli BL21 to express. The optimal expression condition was determined using different concentration of IPTG, rotation speed and time of shaking. For purpose of retain the enzymatic activity, we simplified the steps of purification. We used the ultrasonication and high-speed centrifugation to remove lots of tropina, and salt fractionation to remove hybridprotein, and S-200 sieve chromatography to separate hybridprotein by different molecular weight. HO-1 in vitro activity assay was established to test the activity of HO-1. Second, the antibodies were prepared from the New-Zealand rabbits immunized with purified hHO-1 andΔrHO-1. The titer and specificity of the antibody were determined by ELISA and Western Blot respectively and the neutralization activity of the antibody was determined by HO activity assay. Epitopes ofΔrHO-1 were screened out by Phage Display, and the epitopes sequences were analyzed. The antibodes were prepared from the New-Zealand rabbits immunized with synthesis epitopes peptides. Then test the neutralize activity by HO activity assay.Experimental results:①S equencing of pMW172/hHO-1 plasmid showed the HO-1 gene length was 938bp, and sequence was correct. Then transformed it into E. coli BL21 and expressed soluble hHO-1.②T he purified hHO-1 was obtained by 30%-40% salt fractionation and sieve chromatography and reached to a purity of 90 %, a purification fold of 2.83 and a yield rate of 30.3 %.③T he polyclonal antibody specific to hHO-1 titer was up to 106 by ELISA and had high specificity to hHO-1 tested by Western-Blot. And it can neutralize 46% catalytic activity of hHO-1.④Sequencing of pMW172/ΔrHO-1 plasmid showed the HO-1 gene length was 792bp, and sequence was correct. Then transformed it into E. coli BL21 and expressed solubleΔrHO-1.⑤The purifiedΔrHO-1 was obtained by 35%-55% salt fractionation and sieve chromatography and reached to a purity of 95 %, a purification fold of 1.61 and a yield rate of 34.5 %.⑥The polyclonal antibody specific toΔrHO-1 titer was up to 1.3×107 by ELISA and had high specificity to hHO-1 tested by Western-Blot. And it can neutralize 72.6% catalytic activity ofΔrHO-1.⑦At the same time, we used the antibody ofΔrHO-1 to test the cross reactivity with hHO-1. ELISA and Western-Blot results indicated that antibody ofΔrHO-1 could be used to detect hHO-1. This means that the functional domain of HO-1 is identical.⑧By screening epitopes ofΔrHO-1 by Phage Display, we obtained 12 epitopes.⑨And we got 4 peptides after sequencing: A1, A13, A3 and A7. We decided initially that A1 and A13 peptides sequences were neutralization epitopes by sequence analyzing and numbers of clones. We used synthesized A1, natural peptide and A13 to prepare their polyclonal antibodies.⑩The antibody of A13 can neutralize HO-1 activity, can neutralize 38.7% ofΔrHO-1 catalytic activity.From the results above, we could conclud that: by screening epitopes and verification, A1, natural peptide and A13 peptides were obtained and antibody of A13 can neutralize HO-1 catalytic activity, viz. neutralization epitopes. If its micromolecule single-chain antibody can be synthesized, then it could be used in researches on related diseases and it will provide a solid foundation for clinical diagnosis, therapeutics and prognosis.