血红素加氧酶-1（HO-1）的多抗制备及其抗原表位的筛选
发布时间：2018-08-28 11:33
【摘要】： 血红素加氧酶-1(heme oxygenase-1 ,HO-1)是体内唯一可被诱导的血红素加氧酶,能够降解血红素为等摩尔一氧化碳、胆绿素和铁离子。在多种细胞中,它均能受到很多因素诱导而高表达,例如:血红素、炎症细胞素、加热、重金属、激素、紫外线、缺氧和一氧化氮等,被诱导的HO-1发挥抗炎及调节凋亡的作用。研究表明,HO-1与很多疾病相关,如:肿瘤、肠炎、脓毒血症、氧惊厥、神经元退行性病变、缺血性脑损伤、动脉粥样硬化、哮喘、高血压等。在体内,表达的HO-1抑制内毒素性休克、(组织内)氧过多、急性胸膜炎以及器官移植和缺血-再灌注损伤中的炎症应答,从而在这些条件下,提供有益的帮助。许多证据表明,在很多疾病模型中,HO-1及其产物胆红素/胆绿素能利用其抗炎、抗凋亡、抗增殖及抗氧化特性,来调节其保护作用。HO-1在许多疾病中活性都有提高,因此,对于HO-1在相关疾病中的作用,以及研究HO-1活性变化在相关疾病的诊断、治疗的作用方面,都需要对相关疾病中HO-1活性进行抑制。而中和抗体能中和抗原的活性,即抑制抗原活性。因此,我们需要得到HO-1的中和抗体并筛选出其抗原表位。 我们表达及纯化HO-1,制备其中和抗体,筛选抗原表位,为以后的相关研究、临床诊断、治疗及预后奠定了一定的基础。确定人血红素加氧酶-1(hHO-1)和重组大鼠血红素加氧酶-1(ΔrHO-1)在大肠杆菌中表达条件与纯化方法,并且利用纯化好的蛋白制备中和抗体,利用噬菌体肽展示库筛选出其抗原表位,并制备其多克隆抗体,利用制备好的表位多抗对其进行验证。 首先酶切鉴定原核表达质粒pMW172/hHO-1及pMW172/ΔrHO-1,并对其进行测序,鉴定正确后,转入大肠杆菌BL21,通过改变摇床转速及IPTG浓度确定可溶性HO-1的最佳表达条件。为了避免HO-1的酶活性损失,尽量简化纯化步骤,利用超声波及高速离心去除大量菌体蛋白,又用分级盐析最大限度地保留了HO-1,除去了杂蛋白,S-200柱层析进一步通过分子量差异将杂蛋白分离,通过体外HO-1活性测定方法检测得到HO-1蛋白的活性。之后,利用纯化的HO-1蛋白作为抗原免疫新西兰兔,制备多克隆抗体,利用ELISA方法测定其效价,以及Western-Blot技术检测抗体的特异性,并用HO-1活性测定方法测定抗体的中和活性。利用噬菌体肽展示库技术对HO-1中和型多抗进行筛选,得到HO-1抗原表位,测序后,经序列比对分析,合成可能具有中和活性的抗原表位肽段,免疫新西兰兔,制备其多克隆抗体。进而,通过HO-1体外活性测定方法测定抗体的中和活性,验证其是否具有中和活性的抗原表位。 本课题的实验结果:①原核表达质粒pMW172/hHO-1经测序后,HO-1基因长度为938bp,测序正确。将其转入大肠杆菌BL21后成功表达了可溶性的活性hHO-1。②超声破碎菌体,hHO-1上清经30%-40%盐析纯化及分子筛层析纯化,获得活性hHO-1蛋白,收得率为30.3%,纯化倍数为2.83倍,纯度为90%。③利用纯化后的hHO-1制备出抗hHO-1兔血清,经ELISA测定效价达到106,Western-Blot显示抗体特异性识别hHO-1。对抗体进行中和活性的测定,证实抗体能中和掉46% hHO-1的催化活性。④原核表达质粒pMW172/ΔrHO-1经测序后,HO-1基因长度为792bp,测序正确。将其转入大肠杆菌BL21后成功表达了可溶性的活性ΔrHO-1。⑤超声破碎菌体,ΔrHO-1上清经35%-55%盐析纯化及分子筛层析纯化,获得活性ΔrHO-1蛋白,收得率为34.5%,纯化倍数为1.61倍,纯度为95%。⑥利用纯化后的ΔrHO-1制备出抗ΔrHO-1兔血清,经ELISA测定效价超过1.3×107,Western-Blot显示抗体特异性识别ΔrHO-1。对抗体进行中和活性的测定,证实抗体能中和掉72.6%ΔrHO-1的催化活性。⑦同时,对抗ΔrHO-1多克隆抗体进行了与hHO-1交叉反应性的检测,ELISA及Western-Blot均证实抗ΔrHO-1多抗可以用于hHO-1的检测。即,HO-1功能区是相同的。⑧利用噬菌体肽展示库技术对纯化后抗ΔrHO-1 IgG进行表位筛选,得到12条肽段。⑨对筛选到的12条肽段测序后,得到了四个肽段组:A1组、A13组、A3组以及A7组,经序列比对分析及克隆数目的多少,初步判断A1和A13肽段最可能是中和表位,对A1、A1的天然肽及A13进行合成,制备出肽段多抗。⑩通过验证证实,抗A13的抗体可以中和掉38.7%的ΔrHO-1催化活性,具有中和HO-1活性的功能,为中和表位,A1和天然肽为结合表位。 由以上结果得出如下结论:通过表位筛选及验证,得到了A1、天然肽及A13 3条肽段,其中,A1及其天然肽HO-1的结合表位,A13的抗体具有中和HO-1催化活性的特性,为具有中和HO-1活性的抗原表位。若合成其的小分子单链抗体,则可以应用到相关疾病的研究中,为以后临床诊断、治疗及预后奠定了一定的基础。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
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