FcεR Ⅰα亚基细胞外部分的重组表达单克隆抗体的制备及特异性鉴定

发布时间：2018-08-29 17:50

【摘要】： 过敏性疾病是一种常见疾病,其临床表现有很多种,包括过敏性哮喘、过敏性休克、过敏性紫癜、过敏性鼻炎、过敏性皮炎等。过敏性疾病是由Ⅰ型超敏反应引起的,其主要中间物质为IgE及其高亲和力受体(FcεRⅠ)。过敏原第一次侵入机体,可以诱导B淋巴细胞产生抗原特异性IgE,IgE与靶细胞表面的高亲和力受体结合以后,当相同抗原再次侵入机体时,就可以引发靶细胞内的一系列信号转导,使靶细胞释放组胺等生物活性物质,引起过敏性炎症反应。因此,IgE高亲和力受体是引起过敏性疾病的关键物质,也是本研究的重点对象。引起Ⅰ型超敏反应的物质称为过敏原,我们生活中的一些高蛋白食物、屋尘、花粉、真菌、人与动物皮毛、羽毛、昆虫、寄生虫、药物及其他化学物质等都可作为过敏原,通过吸入、食入、注射或接触使机体致敏。过敏原侵入机体后,鼻咽、扁桃体、支气管、胃肠粘膜等处固有层的浆细胞产生抗原特异性IgE,IgE通过与嗜碱性粒细胞和肥大细胞表面的IgE高亲和力受体(FcεRⅠ)结合,使靶细胞处于致敏状态。 FcεRⅠ是一种由多个亚单位组成的膜糖蛋白,其α亚基分为细胞外区、跨膜区、胞质区三个部分,通过细胞外区的结合位点与IgE结合,是引发过敏反应的首要步骤。α亚基的细胞外区由2个免疫球蛋白样结构(D1、D2)组成,而靠近膜的环状结构是与IgE的Fc段结合的主要部位。α亚基与IgE直接结合的面由D1-D2接口的顶端(即“色氨酸脊”)以及D2域的近接口一侧所组成。FcεRⅠα链结合在IgE Fc段的2个CH3的顶端,且靠近Fc段的对称轴,锲入Fc段的两条链之间。FcεRⅠα链与2个CH3均有CH2-CH3接头(linker)的残基在受体的顶端形成1个不对称的拱形结构。当特异性抗原与靶细胞上两个以上的IgE分子结合,通过桥联反应,使两个以上的IgE分子靠近并发生构型改变。然后,交联的FcεRⅠ可激活两种胞浆内蛋白激酶(Lyn和Syk),使之磷酸化而激活。最后,接头蛋白和GTP交换因子/GTP酶诱导胞内贮存的Ca~(2+)释放。后者再促进细胞脱颗粒、释放血管活性物质及某些酶类,即可出现过敏反应。FcεRⅠ主要表达于嗜碱性粒细胞和肥大细胞,在嗜酸性粒细胞、单核细胞以及血小板表面也有分布,但量较前两种少的多。通过对FcεRⅠα亚基基因的研究发现,人体嗜碱性粒细胞和肥大细胞表面FcεRⅠα亚基的表达受多个转录因子的调节且其C端的表达受体内IgE的影响。J.Kochan从肥大细胞系KU812提取mRNA逆转录得到的cDNA进行了α亚基氨基酸序列的预测,证实,虽然α亚基含有7个N链的糖基化位点,影响受体的分泌和稳定性,但这些糖基化位点对α链的正确折叠是非必须的,且不影响其与IgE的结合。这些为我们重组表达FcεRⅠα亚基细胞外区段提供了有力的支持。单独的α链胞外区可溶性蛋白可与IgE高亲和力结合。Dvid D等发现,去除小鼠的α亚基后,小鼠因不能表达完整的FcεRⅠ而不会发生IgE介导的Ⅰ型超敏反应。国外对过敏性疾病的研究由来已久,利用抗IgE单克隆抗体,或可溶性受体阻断或抑制Ⅰ型超敏反应的发生已成为抗过敏治疗研究的热点,目前已有商品化的抗IgE抗体用于Ⅰ型超敏反应类疾病的治疗,但利用重组人可溶性FcεRⅠα亚基来阻断其与IgE结合的方法尚未在临床上得到运用。国外有研究用哺乳动物细胞和昆虫细胞表达的马FcεRⅠα亚基胞外区可以与马肺泡灌洗液中的IgE结合。孙仁山等也利用重组可溶性FcεRⅠα亚基成功阻断了小鼠被动皮肤过敏反应。鉴于过敏性疾病病人外周血嗜碱性粒细胞含量比正常人增多,且其表面分布的FcεRⅠ量也多,本研究运用RT-PCR方法从过敏性疾病病人外周血嗜碱性粒细胞中成功扩增并克隆出FcεRⅠα亚基细胞外区的基因片段FcεRⅠα,将编码基因FcεRⅠα克隆至pET-28a(+)表达载体,并在大肠杆菌中进行了大量非可溶性表达,用亲和层析法纯化重组蛋白后,用透析复性的方法进行蛋白质复性,免疫小鼠制备单克隆抗体,用ELISA、细胞免疫荧光法鉴定单克隆抗体的特性。结果显示,利用RT-PCR技术成功克隆了编码过敏性疾病IgE高亲和力受体FcεRⅠα亚基胞外区的基因,经测序并与GenBank中序列进行比对,结果完全一致,大小为696bp。并且将编码基因克隆到pET-28a(+)原核表达质粒中,经PCR、限制性酶切分析等鉴定,成功构建了FcεRⅠα-pET28a(+)重组原核表达质粒。重组质粒FcεRⅠα-pET28a(+)在大肠杆菌中表达出重组蛋白,经过表达条件的优化,表达产物主要以非可溶性形式存在。重组蛋白分子量约为23.3kDa,与理论值基本符合,表达量约占菌体总蛋白的30%。利用亲和层析法纯化重组蛋白,纯度达1 5%。用Ni亲和层析柱对蛋白进行纯化,表明该蛋白确实是所要表达的His融合蛋白。进一步用纯化的表达产物免疫小鼠制备免疫血清,ELISA和细胞免疫荧光法分析表明该免疫血清能与重组蛋白反应,而与正常小鼠血清不发生交叉反应,说明重组蛋白具有抗原活性。用纯化的表达产物免疫小鼠制备了12株单抗,以纯化重组蛋白为抗原,12株单抗培养上清的ELISA法检测,OD值为1.628～2.512。细胞免疫荧光显示其中4株能与嗜碱性粒细胞表面的天然蛋白发生特异性反应,与预计情况完全相符。说明4株单抗均为抗FcεRⅠα亚基特异性单抗。以上结果表明,我们已经成功地构建了FcεRⅠα-pET28a(+)原核重组表达质粒,而且在大肠杆菌中大量表达出具有免疫活性的重组蛋白;筛选并建立了多株分泌抗FcεRⅠα亚基的杂交瘤细胞株,为进一步研究该蛋白的功能、在人过敏性疾病的发生及其信号转导、对过敏性疾病的治疗奠定基础。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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