血紅素加氧酶-1（HO-1）的多抗制備及其抗原表位的篩選
發(fā)布時(shí)間：2018-08-28 11:33
【摘要】： 血紅素加氧酶-1(heme oxygenase-1 ,HO-1)是體內(nèi)唯一可被誘導(dǎo)的血紅素加氧酶,能夠降解血紅素為等摩爾一氧化碳、膽綠素和鐵離子。在多種細(xì)胞中,它均能受到很多因素誘導(dǎo)而高表達(dá),例如:血紅素、炎癥細(xì)胞素、加熱、重金屬、激素、紫外線、缺氧和一氧化氮等,被誘導(dǎo)的HO-1發(fā)揮抗炎及調(diào)節(jié)凋亡的作用。研究表明,HO-1與很多疾病相關(guān),如:腫瘤、腸炎、膿毒血癥、氧驚厥、神經(jīng)元退行性病變、缺血性腦損傷、動(dòng)脈粥樣硬化、哮喘、高血壓等。在體內(nèi),表達(dá)的HO-1抑制內(nèi)毒素性休克、(組織內(nèi))氧過多、急性胸膜炎以及器官移植和缺血-再灌注損傷中的炎癥應(yīng)答,從而在這些條件下,提供有益的幫助。許多證據(jù)表明,在很多疾病模型中,HO-1及其產(chǎn)物膽紅素/膽綠素能利用其抗炎、抗凋亡、抗增殖及抗氧化特性,來調(diào)節(jié)其保護(hù)作用。HO-1在許多疾病中活性都有提高,因此,對(duì)于HO-1在相關(guān)疾病中的作用,以及研究HO-1活性變化在相關(guān)疾病的診斷、治療的作用方面,都需要對(duì)相關(guān)疾病中HO-1活性進(jìn)行抑制。而中和抗體能中和抗原的活性,即抑制抗原活性。因此,我們需要得到HO-1的中和抗體并篩選出其抗原表位。 我們表達(dá)及純化HO-1,制備其中和抗體,篩選抗原表位,為以后的相關(guān)研究、臨床診斷、治療及預(yù)后奠定了一定的基礎(chǔ)。確定人血紅素加氧酶-1(hHO-1)和重組大鼠血紅素加氧酶-1(ΔrHO-1)在大腸桿菌中表達(dá)條件與純化方法,并且利用純化好的蛋白制備中和抗體,利用噬菌體肽展示庫篩選出其抗原表位,并制備其多克隆抗體,利用制備好的表位多抗對(duì)其進(jìn)行驗(yàn)證。 首先酶切鑒定原核表達(dá)質(zhì)粒pMW172/hHO-1及pMW172/ΔrHO-1,并對(duì)其進(jìn)行測(cè)序,鑒定正確后,轉(zhuǎn)入大腸桿菌BL21,通過改變搖床轉(zhuǎn)速及IPTG濃度確定可溶性HO-1的最佳表達(dá)條件。為了避免HO-1的酶活性損失,盡量簡(jiǎn)化純化步驟,利用超聲波及高速離心去除大量菌體蛋白,又用分級(jí)鹽析最大限度地保留了HO-1,除去了雜蛋白,S-200柱層析進(jìn)一步通過分子量差異將雜蛋白分離,通過體外HO-1活性測(cè)定方法檢測(cè)得到HO-1蛋白的活性。之后,利用純化的HO-1蛋白作為抗原免疫新西蘭兔,制備多克隆抗體,利用ELISA方法測(cè)定其效價(jià),以及Western-Blot技術(shù)檢測(cè)抗體的特異性,并用HO-1活性測(cè)定方法測(cè)定抗體的中和活性。利用噬菌體肽展示庫技術(shù)對(duì)HO-1中和型多抗進(jìn)行篩選,得到HO-1抗原表位,測(cè)序后,經(jīng)序列比對(duì)分析,合成可能具有中和活性的抗原表位肽段,免疫新西蘭兔,制備其多克隆抗體。進(jìn)而,通過HO-1體外活性測(cè)定方法測(cè)定抗體的中和活性,驗(yàn)證其是否具有中和活性的抗原表位。 本課題的實(shí)驗(yàn)結(jié)果:①原核表達(dá)質(zhì)粒pMW172/hHO-1經(jīng)測(cè)序后,HO-1基因長(zhǎng)度為938bp,測(cè)序正確。將其轉(zhuǎn)入大腸桿菌BL21后成功表達(dá)了可溶性的活性hHO-1。②超聲破碎菌體,hHO-1上清經(jīng)30%-40%鹽析純化及分子篩層析純化,獲得活性hHO-1蛋白,收得率為30.3%,純化倍數(shù)為2.83倍,純度為90%。③利用純化后的hHO-1制備出抗hHO-1兔血清,經(jīng)ELISA測(cè)定效價(jià)達(dá)到106,Western-Blot顯示抗體特異性識(shí)別hHO-1。對(duì)抗體進(jìn)行中和活性的測(cè)定,證實(shí)抗體能中和掉46% hHO-1的催化活性。④原核表達(dá)質(zhì)粒pMW172/ΔrHO-1經(jīng)測(cè)序后,HO-1基因長(zhǎng)度為792bp,測(cè)序正確。將其轉(zhuǎn)入大腸桿菌BL21后成功表達(dá)了可溶性的活性ΔrHO-1。⑤超聲破碎菌體,ΔrHO-1上清經(jīng)35%-55%鹽析純化及分子篩層析純化,獲得活性ΔrHO-1蛋白,收得率為34.5%,純化倍數(shù)為1.61倍,純度為95%。⑥利用純化后的ΔrHO-1制備出抗ΔrHO-1兔血清,經(jīng)ELISA測(cè)定效價(jià)超過1.3×107,Western-Blot顯示抗體特異性識(shí)別ΔrHO-1。對(duì)抗體進(jìn)行中和活性的測(cè)定,證實(shí)抗體能中和掉72.6%ΔrHO-1的催化活性。⑦同時(shí),對(duì)抗ΔrHO-1多克隆抗體進(jìn)行了與hHO-1交叉反應(yīng)性的檢測(cè),ELISA及Western-Blot均證實(shí)抗ΔrHO-1多抗可以用于hHO-1的檢測(cè)。即,HO-1功能區(qū)是相同的。⑧利用噬菌體肽展示庫技術(shù)對(duì)純化后抗ΔrHO-1 IgG進(jìn)行表位篩選,得到12條肽段。⑨對(duì)篩選到的12條肽段測(cè)序后,得到了四個(gè)肽段組:A1組、A13組、A3組以及A7組,經(jīng)序列比對(duì)分析及克隆數(shù)目的多少,初步判斷A1和A13肽段最可能是中和表位,對(duì)A1、A1的天然肽及A13進(jìn)行合成,制備出肽段多抗。⑩通過驗(yàn)證證實(shí),抗A13的抗體可以中和掉38.7%的ΔrHO-1催化活性,具有中和HO-1活性的功能,為中和表位,A1和天然肽為結(jié)合表位。 由以上結(jié)果得出如下結(jié)論:通過表位篩選及驗(yàn)證,得到了A1、天然肽及A13 3條肽段,其中,A1及其天然肽HO-1的結(jié)合表位,A13的抗體具有中和HO-1催化活性的特性,為具有中和HO-1活性的抗原表位。若合成其的小分子單鏈抗體,則可以應(yīng)用到相關(guān)疾病的研究中,為以后臨床診斷、治療及預(yù)后奠定了一定的基礎(chǔ)。
[Abstract]:Heme oxygenase-1 (HO-1) is the only inducible heme oxygenase in the body, which can degrade heme to isomolar carbon monoxide, biliverdin and iron ions. In many cells, it can be induced by many factors and high expression, such as: heme, inflammatory cytokines, heating, heavy metals, hormones, ultraviolet light, hypoxia. HO-1, which is induced by nitric oxide, plays an anti-inflammatory and apoptosis-regulating role. Studies have shown that HO-1 is associated with many diseases, such as tumors, enteritis, sepsis, oxyconvulsions, neurodegenerative diseases, ischemic brain damage, atherosclerosis, asthma, hypertension and so on. Many evidences show that HO-1 and its product bilirubin/biliverdin can modulate the protective effects of HO-1 in many disease models by its anti-inflammatory, anti-apoptotic, anti-proliferative and antioxidant properties. In many diseases, the activity of HO-1 is increased. Therefore, it is necessary to inhibit the activity of HO-1 in the diagnosis and treatment of related diseases. Neutralizing antibodies can neutralize the activity of antigens, that is, inhibit the activity of antigens. Neutralizing antibodies and screening their epitopes.
We expressed and purified HO-1, prepared neutralizing antibodies, screened antigen epitopes, and laid a foundation for future research, clinical diagnosis, treatment and prognosis. The expression conditions and purification methods of human heme oxygenase-1 (hHO-1) and recombinant rat heme oxygenase-1 (rHO-1) in Escherichia coli were determined. The neutralizing antibody was prepared and its antigen epitope was screened out by phage peptide display library. The polyclonal antibody was prepared and verified by the prepared epitope polyantibody.
The prokaryotic expression plasmids pMW172/hHO-1 and pMW172/rHO-1 were identified by enzyme digestion and sequenced. After identification, the plasmids were transferred into E. coli BL21. The optimal expression conditions of soluble HO-1 were determined by changing shaking table speed and IPTG concentration. In order to avoid the loss of HO-1 enzyme activity, the purification steps were simplified as far as possible, and ultrasonic wave and high-speed centrifugation were used to remove them. In addition to a large number of bacterial proteins, HO-1 was retained to the greatest extent by fractional salting out, and the impurity proteins were removed. The impurity proteins were further separated by S-200 column chromatography through molecular weight differences. The activity of HO-1 protein was detected by HO-1 activity assay in vitro. Then, the purified HO-1 protein was used as antigen to immunize New Zealand rabbits to prepare polyclonal antibodies. The titer of the antibody was determined by ELISA, and the specificity of the antibody was detected by Western-Blot technique. The neutralization activity of the antibody was determined by HO-1 activity assay. The polyclonal antibody was prepared by immunizing New Zealand rabbits with antigenic epitope peptides. The neutralization activity of the antibody was determined by HO-1 activity assay in vitro to verify whether the antibody has a neutralizing epitope.
The results of this study were as follows: (1) After the prokaryotic expression plasmid pMW172/hHO-1 was sequenced, the length of HO-1 gene was 938 BP and the sequence was correct. (3) Anti-hHO-1 rabbit serum was prepared by purified hHO-1. The titer of anti-hHO-1 rabbit serum was 106 by ELISA, and the specific recognition of anti-hHO-1 by Western-Blot showed that the antibody could neutralize the catalytic activity of 46% hHO-1. Fourthly, the prokaryotic expression plasmid pMW172/rHO-1 was sequenced. After transfection into E. coli BL21, the soluble active rHO-1._was successfully expressed. The supernatant of rHO-1 was purified by 35% - 55% salt analysis and molecular sieve chromatography, and the active rHO-1 protein was obtained. The yield was 34.5%, the purity was 1.61 times and the purity was 95%. Anti-rHO-1 rabbit serum was prepared and its titer was more than 1.3 *107 by ELISA. Western-Blot showed that the antibody specifically recognized rHO-1. The neutralization activity of the antibody was determined. It was confirmed that the antibody could neutralize the catalytic activity of 72.6% rHO-1. At the same time, the cross-reactivity of anti-rHO-1 polyclonal antibody with hHO-1 was detected, and the cross-reactivity of ELISA and Western-B-HO-1 was detected. Lot confirmed that anti rHO-1 polyclonal antibody could be used for the detection of hHO-1. That is, HO-1 functional region was the same. _The epitope of purified anti rHO-1 IgG was screened by phage display library technology and 12 peptides were obtained. _After sequencing the 12 peptides, four peptides were obtained: A1 group, A13 group, A3 group and A7 group. And the number of clones, preliminary judgment of A1 and A13 peptides are most likely to be neutralizing epitopes, A1, A13 natural peptides and A13 were synthesized, peptide polyantibodies were prepared. _Through verification, anti-A13 antibodies can neutralize 38.7% of the rHO-1 catalytic activity, with the function of neutralizing HO-1 activity, neutralizing epitopes, A1 and natural peptides as binding epitopes.
The results are as follows: A1, natural peptides and A13 3 peptides were obtained by epitope screening and validation. Among them, the binding epitope of A1 and its natural peptide HO-1, and the antibody of A13, which has the characteristics of neutralizing the catalytic activity of HO-1, are antigenic epitopes with the activity of neutralizing HO-1. The study of disease lays a foundation for clinical diagnosis, treatment and prognosis.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
[Abstract]:Heme oxygenase-1 (HO-1) is the only inducible heme oxygenase in the body, which can degrade heme to isomolar carbon monoxide, biliverdin and iron ions. In many cells, it can be induced by many factors and high expression, such as: heme, inflammatory cytokines, heating, heavy metals, hormones, ultraviolet light, hypoxia. HO-1, which is induced by nitric oxide, plays an anti-inflammatory and apoptosis-regulating role. Studies have shown that HO-1 is associated with many diseases, such as tumors, enteritis, sepsis, oxyconvulsions, neurodegenerative diseases, ischemic brain damage, atherosclerosis, asthma, hypertension and so on. Many evidences show that HO-1 and its product bilirubin/biliverdin can modulate the protective effects of HO-1 in many disease models by its anti-inflammatory, anti-apoptotic, anti-proliferative and antioxidant properties. In many diseases, the activity of HO-1 is increased. Therefore, it is necessary to inhibit the activity of HO-1 in the diagnosis and treatment of related diseases. Neutralizing antibodies can neutralize the activity of antigens, that is, inhibit the activity of antigens. Neutralizing antibodies and screening their epitopes.
We expressed and purified HO-1, prepared neutralizing antibodies, screened antigen epitopes, and laid a foundation for future research, clinical diagnosis, treatment and prognosis. The expression conditions and purification methods of human heme oxygenase-1 (hHO-1) and recombinant rat heme oxygenase-1 (rHO-1) in Escherichia coli were determined. The neutralizing antibody was prepared and its antigen epitope was screened out by phage peptide display library. The polyclonal antibody was prepared and verified by the prepared epitope polyantibody.
The prokaryotic expression plasmids pMW172/hHO-1 and pMW172/rHO-1 were identified by enzyme digestion and sequenced. After identification, the plasmids were transferred into E. coli BL21. The optimal expression conditions of soluble HO-1 were determined by changing shaking table speed and IPTG concentration. In order to avoid the loss of HO-1 enzyme activity, the purification steps were simplified as far as possible, and ultrasonic wave and high-speed centrifugation were used to remove them. In addition to a large number of bacterial proteins, HO-1 was retained to the greatest extent by fractional salting out, and the impurity proteins were removed. The impurity proteins were further separated by S-200 column chromatography through molecular weight differences. The activity of HO-1 protein was detected by HO-1 activity assay in vitro. Then, the purified HO-1 protein was used as antigen to immunize New Zealand rabbits to prepare polyclonal antibodies. The titer of the antibody was determined by ELISA, and the specificity of the antibody was detected by Western-Blot technique. The neutralization activity of the antibody was determined by HO-1 activity assay. The polyclonal antibody was prepared by immunizing New Zealand rabbits with antigenic epitope peptides. The neutralization activity of the antibody was determined by HO-1 activity assay in vitro to verify whether the antibody has a neutralizing epitope.
The results of this study were as follows: (1) After the prokaryotic expression plasmid pMW172/hHO-1 was sequenced, the length of HO-1 gene was 938 BP and the sequence was correct. (3) Anti-hHO-1 rabbit serum was prepared by purified hHO-1. The titer of anti-hHO-1 rabbit serum was 106 by ELISA, and the specific recognition of anti-hHO-1 by Western-Blot showed that the antibody could neutralize the catalytic activity of 46% hHO-1. Fourthly, the prokaryotic expression plasmid pMW172/rHO-1 was sequenced. After transfection into E. coli BL21, the soluble active rHO-1._was successfully expressed. The supernatant of rHO-1 was purified by 35% - 55% salt analysis and molecular sieve chromatography, and the active rHO-1 protein was obtained. The yield was 34.5%, the purity was 1.61 times and the purity was 95%. Anti-rHO-1 rabbit serum was prepared and its titer was more than 1.3 *107 by ELISA. Western-Blot showed that the antibody specifically recognized rHO-1. The neutralization activity of the antibody was determined. It was confirmed that the antibody could neutralize the catalytic activity of 72.6% rHO-1. At the same time, the cross-reactivity of anti-rHO-1 polyclonal antibody with hHO-1 was detected, and the cross-reactivity of ELISA and Western-B-HO-1 was detected. Lot confirmed that anti rHO-1 polyclonal antibody could be used for the detection of hHO-1. That is, HO-1 functional region was the same. _The epitope of purified anti rHO-1 IgG was screened by phage display library technology and 12 peptides were obtained. _After sequencing the 12 peptides, four peptides were obtained: A1 group, A13 group, A3 group and A7 group. And the number of clones, preliminary judgment of A1 and A13 peptides are most likely to be neutralizing epitopes, A1, A13 natural peptides and A13 were synthesized, peptide polyantibodies were prepared. _Through verification, anti-A13 antibodies can neutralize 38.7% of the rHO-1 catalytic activity, with the function of neutralizing HO-1 activity, neutralizing epitopes, A1 and natural peptides as binding epitopes.
The results are as follows: A1, natural peptides and A13 3 peptides were obtained by epitope screening and validation. Among them, the binding epitope of A1 and its natural peptide HO-1, and the antibody of A13, which has the characteristics of neutralizing the catalytic activity of HO-1, are antigenic epitopes with the activity of neutralizing HO-1. The study of disease lays a foundation for clinical diagnosis, treatment and prognosis.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李玉明,劉俊昌,張敏,鄭新程,吳立玲,時(shí)安云,伍貽經(jīng);膿毒血癥時(shí)大鼠心血管系統(tǒng)血紅素加氧酶的變化[J];北京醫(yī)科大學(xué)學(xué)報(bào);1999年06期
2 徐建平,練慶林,盧玲,徐偉剛,孫學(xué)軍,蔣春雷;血紅素加氧酶與氧驚厥關(guān)系的研究[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年07期
3 王秀宏,滕悅秋,王志剛,劉明,周虹;血紅素加氧酶在大腸桿菌中穩(wěn)定表達(dá)的氨基酸范圍的研究[J];醫(yī)學(xué)分子生物學(xué)雜志;2004年04期
4 王秀宏,高旭,尹s，
本文編號(hào)：2209270
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2209270.html
最近更新
教材專著
