用基因组DNA剪接技术克隆SIgA相关基因

目的：克隆分泌型IgA（SIgA）相关基因--J链基因(IgJ)、多聚免疫球蛋白受体基因（pIgR）和IgA重链恒定区基因（IGHA）,为进一步构建SIgA真核表达质粒奠定基础。方法：采用本室建立的"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果：PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明本实验获得的上述基因与GenBank中的目标基因序列完全一致。结论：本文通过基因组DNA剪接技术成功克隆人类SIgA三个相关基因,提示此技术是合成多外显子cDNA的有效手段。     

旋毛虫感染小鼠肠道菌群变化的研究

目的通过观察黑龙江株旋毛虫感染小鼠肠道分泌物中分泌型免疫球蛋白A、肠道菌群的变化,探讨感染小鼠肠道菌群的变化。方法分别于小鼠感染黑龙江株旋毛虫后7、14、21、28和35d,观察模型组及对照组小鼠肠道分泌物中的分泌型免疫球蛋白A、肠道双歧杆菌、乳酸杆菌、肠杆菌、肠球菌的菌群变化。sIgA采用放射免疫法检测。结果模型组sIgA分泌水平在感染后14d达高峰,随后缓慢下降但始终保持高水平（P〈0．01）。模型组肠道双歧杆菌的数量在感染后7d略低于对照组（P〈0．05）,第14天降至最低水平,随后逐渐升高,至感染后35d恢复正常水平。乳酸杆菌的数量在感染后7d略低于对照组,第14天降至最低水平,随后逐渐增加（P〈0．05）。肠杆菌的数量在感染后7d略高于对照组,感染后14d明显高于对照组,随后始终保持下降趋势（P〈0．05）。肠球菌在感染后7d略高于对照组（P〈0．05）,在14d明显高于对照组,随后缓慢下降,至感染后35d恢复正常水平。结论旋毛虫感染小鼠sIga的分泌在肠道免疫中发挥重要作用,同时也影响肠道菌群;肠道菌群的变化可能与旋毛虫感染小鼠免疫系统中sIgA的分泌有关。     

抗禽流感病毒H5N1 分泌型IgA 在中国仓鼠卵巢细胞中的表达

分泌型IgA (SIgA) 在机体的粘膜免疫中具有重要作用,在外分泌道中比单体IgA和IgG抗体具有更好的抗感染活性。为了表达抗禽流感病毒H5N1人-鼠嵌合分泌型IgA抗体,首先以本室先前构建的稳定表达IgA的中国仓鼠卵巢细胞 (CHO) 细胞系为基础,共转染分泌片和J链表达质粒,然后用抗生素Zeocin选择阳性克隆细胞,利用倍比稀释的方法筛选分泌SIgA的单克隆细胞,通过Western blotting分析培养上清中SIgA的表达情况。结果表明,在CHO细胞中成功表达了SIgA抗体,上述研究为研制分泌型     

双歧杆菌对SD大鼠空肠黏膜分泌型免疫球蛋白A的影响

目的研究双歧杆菌的免疫佐剂效应。方法将SD大鼠随机分成3组,即卵清白蛋白（OVA）组,双歧杆菌＋OVA组和PBS空白对照组。各组给予免疫后,收集长约3cm的空肠,通过免疫组织化学技术和Qwin图像处理系统,对空肠分泌型免疫球蛋白A（sIgA）的定位进行系统分析,研究同时注射OVA和双歧杆菌后对SD大鼠在不同时期空肠中sIgA阳性细胞分泌的影响。结果在免疫后的SD大鼠的空肠内均产生了特异性的分泌型抗体sIgA,其中双歧杆菌＋OVA组显著高于其他2组（P〈0．05）。结论双歧杆菌经胃肠道黏膜免疫可诱导有效的免疫佐剂效应。     

基因工程抗体的获得

一、抗体结构和抗体基因 1.抗体结构 抗体分为五类,即IgG、IgM、IgA、IgD和IgE,其理化性质各不相同,在体内的比例、分布及代谢速率也很不一样。IgG是最重要的血清免疫球蛋白,分为四种亚型:IgG1是主要的亚型(占IgG总量的67％),其次是IgG2、IgG3、IgG4。IgA是主要的分泌型Ig,分为IgA1,IgA2两个亚型。IgM是一五聚体分子,由10条H链、10条L链和一条J链通过二硫键连接而成。它是一个多价体。在B细胞膜上的IgM作为抗原受体与B细胞成熟、分化有关。     

乳酸杆菌对感染大肠杆菌O157:H7小鼠肠道黏膜免疫的影响

【目的】分析小鼠在感染Escherichia coli O157:H7及补充嗜酸乳杆菌KLDS AD1和瑞士乳杆菌KLDS1.8701期间小肠黏膜中SIgA和细胞因子的变化规律,结合小鼠表象特征,探讨2株乳酸杆菌对小鼠腹泻的治疗效果。【方法】将小鼠分成4组,空白组、致病对照组、嗜酸乳杆菌组和瑞士乳杆菌组,对实验组小鼠连续7 d灌胃大肠杆菌致病后,再连续7 d分别灌胃2株乳酸杆菌,采集小鼠小肠利用ELISA法测得各组小鼠肠道组织中SIgA和4种细胞因子IL-2、IFN-γ、IL-4和IL-6的含量。【结果】瑞士乳杆菌可极显著提高感染大肠杆菌O157:H7小鼠的体重,嗜酸乳杆菌的效果较小;感染E.coli O157:H7后,SIgA、IL-2和IFN-γ的含量在第3天达到最大值,第5天开始下降,而IL-4和IL-6在第5天达到最大值,第7天开始下降。补充嗜酸乳杆菌和瑞士乳杆菌后,SIgA和4种细胞因子的含量都迅速增加,并保持较高水平,与其他两组差异显著。【结论】嗜酸乳杆菌KLDS AD1和瑞士乳杆菌KLDS 1.8701都可通过增加细胞因子和SIgA的分泌增强肠道黏膜免疫,对小鼠腹泻有一定的缓解作用。     

SIgA分泌片分离、纯化研究

为研究SIgA分泌片（Secretory component,SC)分离,纯化及鉴定方法,以SC存在游离和结合两种形式,本研究应用凝胶过滤和盐析法直接从初乳中分离纯化游离SC,并进行鉴定。分离纯化获得蛋白溶液12.4ml,蛋白含量0.85mg/ml,免疫双扩仅与抗SC多克隆抗体（PcAb)反应,分子量约75kD,免疫印迹实验与抗SC单克隆抗体（McAb)和PcAb特异反应,表明纯化蛋白为SC。该SC的分离纯化和鉴定处于不断完善之中,其方法的改进有利于获得更纯的SC,值得粘膜免疫研究者借鉴。     

催化淀粉样蛋白产生的γ分泌酶的组装

γ分泌酶可引起多种膜蛋白的跨膜剪切作用,尤其可导致淀粉样前体蛋白（APP）的跨膜剪切,产生淀粉样蛋白（Aβ）。Aβ易发生沉积而诱发阿尔茨海默氏病（AD）。γ分泌酶由四种组分PS、Aph-1、NCT及Pen-2构成,由于该酶的相对分子质量巨大以及结构复杂,所以研究进展比较缓慢,其结构与功能至今仍未完全揭示。本文概述了在催化Aβ产生时γ分泌酶组装过程的研究进展,包括各组分之间的调控及组装。     

黑木耳种内杂交子同工酶基因座的遗传分析

从黑木耳（Auricularia auricula）种内杂交子H2J3的子实体上单孢分离培养得到F1代52个单核体菌株,采用聚丙烯酰胺凝胶电泳技术对杂交子H2J3的单核体亲本（H2、J3）及F1代52个单核体菌株进行EST、MDH和FDH3个酶系统的同工酶分析。结果表明,黑木耳EST,MDH和FDH3个酶系统分别由7个、5个和4个基因座控制,其多态性基因座分别为4个、1个和0个,其中两个基因座（EST-5,EST-6）之间存在紧密连锁关系。     

贯叶连翘的分泌结构及其与金丝桃素积累的关系

贯叶连翘（ＨｙｐｅｒｉｃｕｍｐｅｒｆｏｒａｔｕｍＬ．）地上器官分布着分泌细胞球（黑色腺点）、分泌囊（半透明腺点）和分泌道（半透明腺条）３类内部分泌结构。分泌细胞球在茎、叶和花器官中均有分布,由２层鞘细胞包围多个分泌细胞构成实心的分泌细胞团。分泌囊主要分布于叶片中,分泌道则分布于花器官中,它们都是由１～２层切向扁平细胞围绕圆形或长形腔道构成,腔道的贮存物为精油。利用组织化学方法,结合荧光显微镜观察,证实金丝桃素类物质是由分泌细胞球（黑色腺点）所合成和积累的。通过用戊二醛和锇酸固定样品的显微和超微结构观察,发现金丝桃素类物质积累在成熟腺体分泌细胞的中央大液泡中,细胞周围浓厚的细胞质中分布着大量小液泡和高尔基体、内质网等细胞器。在此基础上对金丝桃素类物质的积累过程进行了初步探讨     

Assembly, secretion, and vacuolar delivery of a hybrid immunoglobulin in plants

Secretory immunoglobulin (Ig) A is a decameric Ig composed of four alpha-heavy chains, four light chains, a joining (J) chain, and a secretory component (SC). The heavy and light chains form two tetrameric Ig molecules that are joined by the J chain and associate with the SC. Expression of a secretory monoclonal antibody in tobacco (Nicotiana tabacum) has been described: this molecule (secretory IgA/G [SIgA/G]) was modified by having a hybrid heavy chain sequence consisting of IgG gamma-chain domains linked to constant region domains of an IgA alpha-chain. In tobacco, about 70% of the protein assembles to its final, decameric structure. We show here that SIgA/G assembly and secretion are slow, with only approximately 10% of the newly synthesized molecules being secreted after 24 h and the bulk probably remaining in the endoplasmic reticulum. In addition, a proportion of SIgA/G is delivered to the vacuole as at least partially assembled molecules by a process that is blocked by the membrane traffic inhibitor brefeldin A. Neither the SC nor the J chain are responsible for vacuolar delivery, because IgA/G tetramers have the same fate. The parent IgG tetrameric molecule, containing wild-type gamma-heavy chains, is instead secreted rapidly and efficiently. This strongly suggests that intracellular retention and vacuolar delivery of IgA/G is due to the alpha-domains present in the hybrid alpha/gamma-heavy chains and indicates that the plant secretory system may partially deliver to the vacuole recombinant proteins expected to be secreted.     

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.     

Some Properties of Secretory IgA Purified from Bovine Colostrum

The major component of a purified sample of secretory IgA (SIgA) in colostrum was revealed as a single peak on gel filtration with Sepharose 6B, having an estimated molecular weight of 540,000. The existence of a higher molecular weight component was suggested by a small shoulder on the ascending limb of the peak, but another component of IgA reported as IgA lacking the secretory component (SC) could not be found. When the purified SIgA was concentrated by dialysis against polyethylene glycol, its molecular size was apparently significantly decreased.

Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) showed that all SC in SIgA binds covalently. The band corresponding to the J chain was easily detected when a reduced and alkylated sample was analysed. Estimation of the molecular weight by SDS-PAGE gave the following values for each of the constituent polypeptide chains of bovine colostral SIgA: SC, 76,000; H chain, 62,000; L chain, 23,000; and J chain, 18,000. The molecular weight of the whole molecule was calculated to be 434,000.

Analysis of carbohydrates by gas-liquid chromatography showed 6.8% neutral and amino hexoses, consisting of 0.4% fucose, 1.8% mannose, 1.1% galactose and 3.5% glucosamine. Galactosamine, which has been found in bovine free secretory component from milk, could not be detected.     

Secretory IgA N- and O-glycans provide a link between the innate and adaptive immune systems

Secretory IgA (SIgA) is a multi-polypeptide complex consisting of a secretory component (SC) covalently attached to dimeric IgA containing one joining (J) chain. We present the analysis of both the N- and O-glycans on the individual peptides from this complex. Based on these data, we have constructed a molecular model of SIgA1 with all its glycans, in which the Fab arms form a T shape and the SC is wrapped around the heavy chains. The O-glycan regions on the heavy (H) chains and the SC N-glycans have adhesin-binding glycan epitopes including galactose-linked beta1-4 and beta1-3 to GlcNAc, fucose-linked alpha1-3 and alpha1-4 to GlcNAc and alpha1-2 to galactose, and alpha2-3 and alpha2-6-linked sialic acids. These glycan epitopes provide SIgA with further bacteria-binding sites in addition to the four Fab-binding sites, thus enabling SIgA to participate in both innate and adaptive immunity. We also show that the N-glycans on the H chains of both SIgA1 and SIgA2 present terminal GlcNAc and mannose residues that are normally masked by SC, but that can be unmasked and recognized by mannose-binding lectin, by disrupting the SC-H chain noncovalent interactions.     

Simplified procedure to recover recombinant antigenized secretory IgA to be used as a vaccine vector

Induced protection mechanisms at mucosal surfaces involve secretory IgA (SIgA), a complex structure made of polymeric-dimeric IgA (IgA(p/d)) antibody associated with secretory component (SC). SIgA can adhere to M cells of the intestinal and nasal epithelia, are transported across these latter, and are thus available to the immune cells underlying the epithelia. This property makes SIgA suitable as potential mucosal vaccine delivery vector. It remains that production and purification of SIgA is a complex task since IgA(p/d) and SC are naturally synthesized by two different cell types. Furthermore, only IgA(p/d) are capable to associate with SC. Thus, we sought to separate IgA(p/d) and monomeric IgA (IgA(m)) antibodies secreted by hybridoma cells in CELLine bioreactors. To this aim, we connected together two 1-m long columns filled with Sephacryl S-300 beads and placed them under the control of a automatized chromatographic system. In parallel, we produced recombinant antigenized human SC (ra-hSC) in Chinese hamster ovary (CHO) cells adapted to suspension culture in CELLine bioreactors. To avoid intermediate purification of ra-hSC, culture supernatants (SN) containing this latter were combined with purified IgA(p/d), and the recombinant antigenized SIgA (raSIgA) complex was resolved on a 1-m long column filled with Superdex 200 beads. Biochemical characterization based on SDS-PAGE, silver staining, immunodetection and enzyme-linked immunosorbent assay (ELISA) indicates that highly purified raSIgA can be recovered using this simple two-step procedure. Such preparations are currently used to immunize mice to induce mucosal and systemic responses.     

Method of preparation and evaluation of antisera for the differential determination of secretory immunoglobulin A and free secretory component in biological fluids]

One of the pressing tasks in the study of local nonsusceptibility to infectious diseases and immunochemical analysis of the external secretion is recording of the level of various forms of the secretory IgA (SIgA) and of the secretory component (SC) in various biological fluids. Indication and measurment of the concentrations of the mentioned proteins encounter serious difficulties caused by heterogeneity of their molecular forms. It was shown that the antisera to the whole molecule of SIgA and SC are of no use. On the basis of a new method of purification of free SC and technology of preparation of monospecific antisera capable of separation of SIgA and free SC there were obtained diagnostic antisera for the quantitative recording and differentiation of various forms of IgA and SC in biological fluids. A reliable measurement of the SIgA and SC concentration in some external secretion was carried out with the aid of the mentioned preparations without any complicated chromatographic experiments.     

Peculiar secretory IgA system identified in chickens. II. Identification and distribution of free secretory component and immunoglobulins of IgA, IgM, and IgG in chicken external secretions.

A homologue of a free secretory component (SC) was identified in chicken intestinal secretion by criteria based on its antigenic relationship with intestinal secretory IgA (SIgA), molecular size, sugar content, and electrophoretic mobility, as well as its elution characteristic from ion-exchange chromatography. SC was obtained in a form free from IgA from the intestinal secretion by salting out and DEAE chromatography, followed by density ultracentrifuguation or Sephadex G-200 gel-filtration. However, the free SC revealed some antigenic deficiency when compared to bound SC of intestinal SIgA and showed a failure of binding to serum-type-polymeric IgA of biliary IgA in vitro. Several kinds of chicken external secretions were examined for detection of SC and immunoglobulin classes of IgG, IgA, and IgM. In spite of the wide distribution of immunoglobulins in the external secretions, SC antigen could be detected only in intestinal secretion. Most IgA in the secretions had a molecular structure of a tetramer of serum-type IgA, lacking in SC and having 17S to 18.5S and 600,000 to 700,000 daltons. On the other hand, IgA in the intestinal secretion showed close similarity to the mammalian SIgA, associated with SC and having 11.2S and 350,000 daltons. Presence of antibody activity in the intestinal IgA to avian reovirus was confirmed by plaque reduction tests.     

Effect of a disulfide-interchange enzyme on the assembly of human secretory immunoglobulin A from immunoglobulin A and free secretory component.

A disulfide-interchange enzyme from rat liver microsomes was found to promote binding in vitro of human free secretory component (SC) to dimeric serum-type IgA containing J chain, as assessed by immune precipitation and gel filtration. This effect was greater withe native than with partially reduced SC. Most of the bound SC was covalently linked, as determined by electrophoresis in polyacrylamide gels in detergent. The enzyme did not promote binding of native or partially reduce SC to IgG, IgA monomer, IgA dimer without J chain, or IgM. In the case of IgM, the enzyme did, however, promote covalent bonding of previously non-covalently linked SC. The results overall suggest that a disulfide-interchange enzyme could play a role in vivo in the cell-associated assembly of secretory IgA by promoting the covalent attachment of SC to a dimer of serum-type IgA and that the J chain in the IgA dimer contributes to the enzyme effect.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.