FcεR Ⅰα亞基細(xì)胞外部分的重組表達(dá)單克隆抗體的制備及特異性鑒定
發(fā)布時(shí)間：2018-08-29 17:50
【摘要】： 過(guò)敏性疾病是一種常見(jiàn)疾病,其臨床表現(xiàn)有很多種,包括過(guò)敏性哮喘、過(guò)敏性休克、過(guò)敏性紫癜、過(guò)敏性鼻炎、過(guò)敏性皮炎等。過(guò)敏性疾病是由Ⅰ型超敏反應(yīng)引起的,其主要中間物質(zhì)為IgE及其高親和力受體(FcεRⅠ)。過(guò)敏原第一次侵入機(jī)體,可以誘導(dǎo)B淋巴細(xì)胞產(chǎn)生抗原特異性IgE,IgE與靶細(xì)胞表面的高親和力受體結(jié)合以后,當(dāng)相同抗原再次侵入機(jī)體時(shí),就可以引發(fā)靶細(xì)胞內(nèi)的一系列信號(hào)轉(zhuǎn)導(dǎo),使靶細(xì)胞釋放組胺等生物活性物質(zhì),引起過(guò)敏性炎癥反應(yīng)。因此,IgE高親和力受體是引起過(guò)敏性疾病的關(guān)鍵物質(zhì),也是本研究的重點(diǎn)對(duì)象。 引起Ⅰ型超敏反應(yīng)的物質(zhì)稱為過(guò)敏原,我們生活中的一些高蛋白食物、屋塵、花粉、真菌、人與動(dòng)物皮毛、羽毛、昆蟲(chóng)、寄生蟲(chóng)、藥物及其他化學(xué)物質(zhì)等都可作為過(guò)敏原,通過(guò)吸入、食入、注射或接觸使機(jī)體致敏。過(guò)敏原侵入機(jī)體后,鼻咽、扁桃體、支氣管、胃腸粘膜等處固有層的漿細(xì)胞產(chǎn)生抗原特異性IgE,IgE通過(guò)與嗜堿性粒細(xì)胞和肥大細(xì)胞表面的IgE高親和力受體(FcεRⅠ)結(jié)合,使靶細(xì)胞處于致敏狀態(tài)。 FcεRⅠ是一種由多個(gè)亞單位組成的膜糖蛋白,其α亞基分為細(xì)胞外區(qū)、跨膜區(qū)、胞質(zhì)區(qū)三個(gè)部分,通過(guò)細(xì)胞外區(qū)的結(jié)合位點(diǎn)與IgE結(jié)合,是引發(fā)過(guò)敏反應(yīng)的首要步驟。α亞基的細(xì)胞外區(qū)由2個(gè)免疫球蛋白樣結(jié)構(gòu)(D1、D2)組成,而靠近膜的環(huán)狀結(jié)構(gòu)是與IgE的Fc段結(jié)合的主要部位。α亞基與IgE直接結(jié)合的面由D1-D2接口的頂端(即“色氨酸脊”)以及D2域的近接口一側(cè)所組成。FcεRⅠα鏈結(jié)合在IgE Fc段的2個(gè)CH3的頂端,且靠近Fc段的對(duì)稱軸,鍥入Fc段的兩條鏈之間。FcεRⅠα鏈與2個(gè)CH3均有CH2-CH3接頭(linker)的殘基在受體的頂端形成1個(gè)不對(duì)稱的拱形結(jié)構(gòu)。當(dāng)特異性抗原與靶細(xì)胞上兩個(gè)以上的IgE分子結(jié)合,通過(guò)橋聯(lián)反應(yīng),使兩個(gè)以上的IgE分子靠近并發(fā)生構(gòu)型改變。然后,交聯(lián)的FcεRⅠ可激活兩種胞漿內(nèi)蛋白激酶(Lyn和Syk),使之磷酸化而激活。最后,接頭蛋白和GTP交換因子/GTP酶誘導(dǎo)胞內(nèi)貯存的Ca~(2+)釋放。后者再促進(jìn)細(xì)胞脫顆粒、釋放血管活性物質(zhì)及某些酶類,即可出現(xiàn)過(guò)敏反應(yīng)。FcεRⅠ主要表達(dá)于嗜堿性粒細(xì)胞和肥大細(xì)胞,在嗜酸性粒細(xì)胞、單核細(xì)胞以及血小板表面也有分布,但量較前兩種少的多。 通過(guò)對(duì)FcεRⅠα亞基基因的研究發(fā)現(xiàn),人體嗜堿性粒細(xì)胞和肥大細(xì)胞表面FcεRⅠα亞基的表達(dá)受多個(gè)轉(zhuǎn)錄因子的調(diào)節(jié)且其C端的表達(dá)受體內(nèi)IgE的影響。J.Kochan從肥大細(xì)胞系KU812提取mRNA逆轉(zhuǎn)錄得到的cDNA進(jìn)行了α亞基氨基酸序列的預(yù)測(cè),證實(shí),雖然α亞基含有7個(gè)N鏈的糖基化位點(diǎn),影響受體的分泌和穩(wěn)定性,但這些糖基化位點(diǎn)對(duì)α鏈的正確折疊是非必須的,且不影響其與IgE的結(jié)合。這些為我們重組表達(dá)FcεRⅠα亞基細(xì)胞外區(qū)段提供了有力的支持。單獨(dú)的α鏈胞外區(qū)可溶性蛋白可與IgE高親和力結(jié)合。Dvid D等發(fā)現(xiàn),去除小鼠的α亞基后,小鼠因不能表達(dá)完整的FcεRⅠ而不會(huì)發(fā)生IgE介導(dǎo)的Ⅰ型超敏反應(yīng)。 國(guó)外對(duì)過(guò)敏性疾病的研究由來(lái)已久,利用抗IgE單克隆抗體,或可溶性受體阻斷或抑制Ⅰ型超敏反應(yīng)的發(fā)生已成為抗過(guò)敏治療研究的熱點(diǎn),目前已有商品化的抗IgE抗體用于Ⅰ型超敏反應(yīng)類疾病的治療,但利用重組人可溶性FcεRⅠα亞基來(lái)阻斷其與IgE結(jié)合的方法尚未在臨床上得到運(yùn)用。國(guó)外有研究用哺乳動(dòng)物細(xì)胞和昆蟲(chóng)細(xì)胞表達(dá)的馬FcεRⅠα亞基胞外區(qū)可以與馬肺泡灌洗液中的IgE結(jié)合。孫仁山等也利用重組可溶性FcεRⅠα亞基成功阻斷了小鼠被動(dòng)皮膚過(guò)敏反應(yīng)。 鑒于過(guò)敏性疾病病人外周血嗜堿性粒細(xì)胞含量比正常人增多,且其表面分布的FcεRⅠ量也多,本研究運(yùn)用RT-PCR方法從過(guò)敏性疾病病人外周血嗜堿性粒細(xì)胞中成功擴(kuò)增并克隆出FcεRⅠα亞基細(xì)胞外區(qū)的基因片段FcεRⅠα,將編碼基因FcεRⅠα克隆至pET-28a(+)表達(dá)載體,并在大腸桿菌中進(jìn)行了大量非可溶性表達(dá),用親和層析法純化重組蛋白后,用透析復(fù)性的方法進(jìn)行蛋白質(zhì)復(fù)性,免疫小鼠制備單克隆抗體,用ELISA、細(xì)胞免疫熒光法鑒定單克隆抗體的特性。 結(jié)果顯示,利用RT-PCR技術(shù)成功克隆了編碼過(guò)敏性疾病IgE高親和力受體FcεRⅠα亞基胞外區(qū)的基因,經(jīng)測(cè)序并與GenBank中序列進(jìn)行比對(duì),結(jié)果完全一致,大小為696bp。并且將編碼基因克隆到pET-28a(+)原核表達(dá)質(zhì)粒中,經(jīng)PCR、限制性酶切分析等鑒定,成功構(gòu)建了FcεRⅠα-pET28a(+)重組原核表達(dá)質(zhì)粒。 重組質(zhì)粒FcεRⅠα-pET28a(+)在大腸桿菌中表達(dá)出重組蛋白,經(jīng)過(guò)表達(dá)條件的優(yōu)化,表達(dá)產(chǎn)物主要以非可溶性形式存在。重組蛋白分子量約為23.3kDa,與理論值基本符合,表達(dá)量約占菌體總蛋白的30%。利用親和層析法純化重組蛋白,純度達(dá)1 5%。用Ni親和層析柱對(duì)蛋白進(jìn)行純化,表明該蛋白確實(shí)是所要表達(dá)的His融合蛋白。進(jìn)一步用純化的表達(dá)產(chǎn)物免疫小鼠制備免疫血清,ELISA和細(xì)胞免疫熒光法分析表明該免疫血清能與重組蛋白反應(yīng),而與正常小鼠血清不發(fā)生交叉反應(yīng),說(shuō)明重組蛋白具有抗原活性。 用純化的表達(dá)產(chǎn)物免疫小鼠制備了12株單抗,以純化重組蛋白為抗原,12株單抗培養(yǎng)上清的ELISA法檢測(cè),OD值為1.628～2.512。細(xì)胞免疫熒光顯示其中4株能與嗜堿性粒細(xì)胞表面的天然蛋白發(fā)生特異性反應(yīng),與預(yù)計(jì)情況完全相符。說(shuō)明4株單抗均為抗FcεRⅠα亞基特異性單抗。 以上結(jié)果表明,我們已經(jīng)成功地構(gòu)建了FcεRⅠα-pET28a(+)原核重組表達(dá)質(zhì)粒,而且在大腸桿菌中大量表達(dá)出具有免疫活性的重組蛋白;篩選并建立了多株分泌抗FcεRⅠα亞基的雜交瘤細(xì)胞株,為進(jìn)一步研究該蛋白的功能、在人過(guò)敏性疾病的發(fā)生及其信號(hào)轉(zhuǎn)導(dǎo)、對(duì)過(guò)敏性疾病的治療奠定基礎(chǔ)。
[Abstract]:Allergic disease is a common disease with many clinical manifestations, including allergic asthma, allergic shock, allergic purpura, allergic rhinitis, allergic dermatitis and so on. Allergic disease is caused by type I hypersensitivity, the main intermediate is IgE and its high affinity receptor (Fc epsilon R I). Allergens first invade the body, may In order to induce B lymphocytes to produce antigen-specific IgE, IgE binds to high affinity receptors on the surface of target cells. When the same antigen invades the body again, it can trigger a series of signal transduction in target cells, so that target cells release histamine and other bioactive substances, causing allergic inflammation. The key substance of allergic diseases is also the focus of this study.
The substances that cause type I hypersensitivity are called allergens. Some high protein foods in our lives, house dust, pollen, fungi, human and animal fur, feathers, insects, parasites, drugs and other chemicals can be used as allergens to sensitize the body by inhalation, ingestion, injection or contact. Plasma cells in the lamina propria of the bronchi and gastrointestinal mucosa produce antigen-specific IgE, which binds to the high affinity receptor (Fc epsilon R I) on the surface of basophils and mast cells to sensitize the target cells.
Fc epsilon RI is a membrane glycoprotein composed of several subunits. Its alpha subunit is divided into three parts: extracellular region, transmembrane region and cytoplasmic region. The binding site of the extracellular region to IgE is the first step to induce allergic reaction. The extracellular region of the alpha subunit is composed of two immunoglobulin-like structures (D1, D2) and the ring structure near the membrane. Fc epsilon RI alpha chain is bound to the top of two CH3 segments of the IgE Fc segment and is close to the symmetrical axis of the Fc segment. The residues of the 3-junction (linker) form an asymmetric arched structure at the top of the receptor. When the specific antigen binds to more than two IgE molecules on the target cell, the bridging reaction causes two or more IgE molecules to approach and change their configuration. Then, the cross-linked Fc epsilon RI activates two intracytoplasmic protein kinases (Lyn and Syk) to phosphorylate them. Finally, adaptor proteins and GTP-exchanger/GTP enzymes induce the release of Ca~ (2+) from intracellular storage. The latter promotes cell degranulation, releases vasoactive substances and certain enzymes, resulting in anaphylaxis. Fc epsilon RI is mainly expressed in basophils and mast cells, in eosinophils, monocytes and on platelet surface. The surface is also distributed, but it is much less than the first two.
It was found that the expression of Fc epsilon R I alpha subunit on the surface of human basophils and mast cells was regulated by multiple transcription factors and influenced by IgE in the C-terminal expression receptor. The amino acid sequence of the alpha subunit was predicted by J. Kochan's reverse transcription cDNA from the mRNA of mast cell line KU812. Although the alpha subunit contains seven N-chain glycosylation sites that affect the secretion and stability of the receptor, these glycosylation sites are not necessary for the correct folding of the alpha chain and do not affect its binding to IgE. Dvid D et al found that after removing the alpha subunit of the mice, the mice could not express the complete Fc epsilon RI and could not produce IgE-mediated type I hypersensitivity.
Overseas studies on allergic diseases have a long history. The use of anti-IgE monoclonal antibodies or soluble receptors to block or inhibit the occurrence of type I hypersensitivity has become a hot spot in anti-allergic therapy. At present, commercial anti-IgE antibodies have been used in the treatment of type I hypersensitivity diseases, but recombinant human soluble Fc epsilon R I alpha subunit has been used. Extracellular domain of equine Fc epsilon RI alpha expressed in mammalian and insect cells can bind to IgE in equine alveolar lavage fluid. Sun Renshan et al successfully blocked passive skin hypersensitivity in mice by recombinant soluble Fc epsilon RI alpha subunit.
In view of the fact that the content of basophils in peripheral blood of patients with allergic diseases is higher than that of normal people and the amount of Fc epsilon RI on the surface of patients with allergic diseases is more than that of normal people, the gene fragment Fc epsilon RI alpha encoding Fc epsilon RI alpha subunit was successfully amplified and cloned from basophils of peripheral blood of patients with allergic diseases by RT-PCR. The recombinant protein was purified by affinity chromatography and renatured by dialysis. The monoclonal antibody was prepared by immunizing mice. The characteristics of the monoclonal antibody were identified by ELISA and cell immunofluorescence.
The results showed that the gene encoding the extracellular domain of the high affinity receptor Fc epsilon RI alpha subunit of IgE in allergic diseases was successfully cloned by RT-PCR. The results were identical with those in GenBank. The gene was cloned into the prokaryotic expression plasmid of pET-28a(+) and identified by PCR and restriction enzyme digestion analysis. The Recombinant Prokaryotic Expression Plasmid of Fc e R I -pET28a (+) was successfully constructed.
The recombinant plasmid Fc epsilon R I alpha-pET28a (+) was expressed in E. coli. The recombinant protein was mainly expressed in non-soluble form. The molecular weight of the recombinant protein was about 23.3 kDa, which accounted for about 30% of the total bacterial protein. The purity of the recombinant protein was 15% by affinity chromatography. The protein was purified by Ni affinity chromatography column, which showed that the protein was indeed the fusion protein of His. Immune serum was prepared by immunizing mice with the purified expression product. ELISA and cytoimmunofluorescence analysis showed that the immune serum could react with the recombinant protein, but did not cross-react with the normal mice serum, indicating that it was heavy. Histone has antigen activity.
Twelve monoclonal antibodies were prepared by immunizing mice with purified expressed products. The purified recombinant proteins were used as antigens and the supernatants of 12 monoclonal antibodies were detected by ELISA. The OD values were 1.628-2.512. Cell immunofluorescence showed that four of them could react specifically with the natural proteins on the surface of basophils, which was in good agreement with the predicted results. It is an anti Fc epsilon R I alpha subunit specific monoclonal antibody.
These results indicate that we have successfully constructed Fc epsilon RI alpha-pET28a (+) Prokaryotic Recombinant Expression plasmid, and expressed a large number of recombinant proteins with immune activity in E. coli; screened and established a number of hybridoma cell lines secreting anti-Fc epsilon RI alpha subunit, in order to further study the function of the protein in human allergic diseases. Occurrence and signal transduction provide a basis for the treatment of allergic diseases.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
本文編號(hào)：2211957
[Abstract]:Allergic disease is a common disease with many clinical manifestations, including allergic asthma, allergic shock, allergic purpura, allergic rhinitis, allergic dermatitis and so on. Allergic disease is caused by type I hypersensitivity, the main intermediate is IgE and its high affinity receptor (Fc epsilon R I). Allergens first invade the body, may In order to induce B lymphocytes to produce antigen-specific IgE, IgE binds to high affinity receptors on the surface of target cells. When the same antigen invades the body again, it can trigger a series of signal transduction in target cells, so that target cells release histamine and other bioactive substances, causing allergic inflammation. The key substance of allergic diseases is also the focus of this study.
The substances that cause type I hypersensitivity are called allergens. Some high protein foods in our lives, house dust, pollen, fungi, human and animal fur, feathers, insects, parasites, drugs and other chemicals can be used as allergens to sensitize the body by inhalation, ingestion, injection or contact. Plasma cells in the lamina propria of the bronchi and gastrointestinal mucosa produce antigen-specific IgE, which binds to the high affinity receptor (Fc epsilon R I) on the surface of basophils and mast cells to sensitize the target cells.
Fc epsilon RI is a membrane glycoprotein composed of several subunits. Its alpha subunit is divided into three parts: extracellular region, transmembrane region and cytoplasmic region. The binding site of the extracellular region to IgE is the first step to induce allergic reaction. The extracellular region of the alpha subunit is composed of two immunoglobulin-like structures (D1, D2) and the ring structure near the membrane. Fc epsilon RI alpha chain is bound to the top of two CH3 segments of the IgE Fc segment and is close to the symmetrical axis of the Fc segment. The residues of the 3-junction (linker) form an asymmetric arched structure at the top of the receptor. When the specific antigen binds to more than two IgE molecules on the target cell, the bridging reaction causes two or more IgE molecules to approach and change their configuration. Then, the cross-linked Fc epsilon RI activates two intracytoplasmic protein kinases (Lyn and Syk) to phosphorylate them. Finally, adaptor proteins and GTP-exchanger/GTP enzymes induce the release of Ca~ (2+) from intracellular storage. The latter promotes cell degranulation, releases vasoactive substances and certain enzymes, resulting in anaphylaxis. Fc epsilon RI is mainly expressed in basophils and mast cells, in eosinophils, monocytes and on platelet surface. The surface is also distributed, but it is much less than the first two.
It was found that the expression of Fc epsilon R I alpha subunit on the surface of human basophils and mast cells was regulated by multiple transcription factors and influenced by IgE in the C-terminal expression receptor. The amino acid sequence of the alpha subunit was predicted by J. Kochan's reverse transcription cDNA from the mRNA of mast cell line KU812. Although the alpha subunit contains seven N-chain glycosylation sites that affect the secretion and stability of the receptor, these glycosylation sites are not necessary for the correct folding of the alpha chain and do not affect its binding to IgE. Dvid D et al found that after removing the alpha subunit of the mice, the mice could not express the complete Fc epsilon RI and could not produce IgE-mediated type I hypersensitivity.
Overseas studies on allergic diseases have a long history. The use of anti-IgE monoclonal antibodies or soluble receptors to block or inhibit the occurrence of type I hypersensitivity has become a hot spot in anti-allergic therapy. At present, commercial anti-IgE antibodies have been used in the treatment of type I hypersensitivity diseases, but recombinant human soluble Fc epsilon R I alpha subunit has been used. Extracellular domain of equine Fc epsilon RI alpha expressed in mammalian and insect cells can bind to IgE in equine alveolar lavage fluid. Sun Renshan et al successfully blocked passive skin hypersensitivity in mice by recombinant soluble Fc epsilon RI alpha subunit.
In view of the fact that the content of basophils in peripheral blood of patients with allergic diseases is higher than that of normal people and the amount of Fc epsilon RI on the surface of patients with allergic diseases is more than that of normal people, the gene fragment Fc epsilon RI alpha encoding Fc epsilon RI alpha subunit was successfully amplified and cloned from basophils of peripheral blood of patients with allergic diseases by RT-PCR. The recombinant protein was purified by affinity chromatography and renatured by dialysis. The monoclonal antibody was prepared by immunizing mice. The characteristics of the monoclonal antibody were identified by ELISA and cell immunofluorescence.
The results showed that the gene encoding the extracellular domain of the high affinity receptor Fc epsilon RI alpha subunit of IgE in allergic diseases was successfully cloned by RT-PCR. The results were identical with those in GenBank. The gene was cloned into the prokaryotic expression plasmid of pET-28a(+) and identified by PCR and restriction enzyme digestion analysis. The Recombinant Prokaryotic Expression Plasmid of Fc e R I -pET28a (+) was successfully constructed.
The recombinant plasmid Fc epsilon R I alpha-pET28a (+) was expressed in E. coli. The recombinant protein was mainly expressed in non-soluble form. The molecular weight of the recombinant protein was about 23.3 kDa, which accounted for about 30% of the total bacterial protein. The purity of the recombinant protein was 15% by affinity chromatography. The protein was purified by Ni affinity chromatography column, which showed that the protein was indeed the fusion protein of His. Immune serum was prepared by immunizing mice with the purified expression product. ELISA and cytoimmunofluorescence analysis showed that the immune serum could react with the recombinant protein, but did not cross-react with the normal mice serum, indicating that it was heavy. Histone has antigen activity.
Twelve monoclonal antibodies were prepared by immunizing mice with purified expressed products. The purified recombinant proteins were used as antigens and the supernatants of 12 monoclonal antibodies were detected by ELISA. The OD values were 1.628-2.512. Cell immunofluorescence showed that four of them could react specifically with the natural proteins on the surface of basophils, which was in good agreement with the predicted results. It is an anti Fc epsilon R I alpha subunit specific monoclonal antibody.
These results indicate that we have successfully constructed Fc epsilon RI alpha-pET28a (+) Prokaryotic Recombinant Expression plasmid, and expressed a large number of recombinant proteins with immune activity in E. coli; screened and established a number of hybridoma cell lines secreting anti-Fc epsilon RI alpha subunit, in order to further study the function of the protein in human allergic diseases. Occurrence and signal transduction provide a basis for the treatment of allergic diseases.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 楊丹榕,修清玉,韓煥興;人FcεRIα亞基細(xì)胞外區(qū)的原核表達(dá)及其和IgE結(jié)合的機(jī)制[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2003年11期
2 李莉,李先興,徐銀海,張玲珍,孔憲濤,仲人前;膜結(jié)合型抗大鼠FcεRIα單克隆抗體制備及活性鑒定[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2004年12期
3 吉清,何鳳田;包涵體復(fù)性的研究進(jìn)展[J];國(guó)外醫(yī)學(xué).臨床生物化學(xué)與檢驗(yàn)學(xué)分冊(cè);2004年06期
4 郭克泰;FcεRⅠ信號(hào)傳導(dǎo)途徑與過(guò)敏性疾病的治療[J];國(guó)外醫(yī)學(xué)(免疫學(xué)分冊(cè));2002年05期
5 孫仁山,陳曉紅,冉新澤,程天民,劉榮卿;重組可溶性IgE受體對(duì)小鼠被動(dòng)皮膚過(guò)敏反應(yīng)的抑制效應(yīng)[J];中國(guó)麻風(fēng)皮膚病雜志;2005年11期
6 馮小黎;重組包涵體蛋白質(zhì)的折疊復(fù)性[J];生物化學(xué)與生物物理進(jìn)展;2001年04期
7 寧云山,李妍,王小寧;包含體蛋白質(zhì)的復(fù)性研究進(jìn)展[J];生物技術(shù)通訊;2001年03期
8 王曉川;;兒童過(guò)敏性疾病診斷的幾個(gè)問(wèn)題[J];實(shí)用兒科臨床雜志;2007年09期
9 朱薇,何韶衡,林梓霞,傅意玲;人嗜堿性粒細(xì)胞在不同刺激物作用下的組胺釋放能力[J];細(xì)胞與分子免疫學(xué)雜志;2005年04期
10 張玉環(huán);免疫與變態(tài)反應(yīng)性疾病[J];浙江中西醫(yī)結(jié)合雜志;2005年03期
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