乙肝免疫球蛋白抗体的多样性

目的 基于重链可变区的编码基因序列对乙肝免疫球蛋白（HBIG）中的IgG抗体进行分类。方法 借用文献提供的引物,以HBsAb强阳性健康个体外周血中的总RNA为模板进行RT-PCR和Nested-PCR扩增,通过将产物克隆到T载体、随机挑选测序,分析并统计出插入子的V_H-J_H-C_γ组
合方式。结果 共获得56个有效克隆,全部为人V_H3-D-J_H-C_γ序列,可分成49种序列,其中5种序列有2或3个序列完全相同的克隆。4个Cγ功能基因也全部出现,其中IGHG2频率最高（28次）;23个V_H3功能基因有11个出现,其中频率最高的是IGHV3-23（29次）;23个D功能基因有16个出
现,其中频率最高的是IGHD1-26（8次）;6个J_H 功能基因则全部出现,其中频率最高的是IGHJ4（33次）。V_H3-D-J_H组合有33种,频率最高的是IGHV3-23/IGHD1-26/IGHJ4（8次）,其中5种与IGHG2拼接,但这5种V_H3-D-J_H-C_γ2序列仍有明显的差异。结论 成功地从HBsAb强阳性健康个体外周血中克隆到由VH3亚家族参与重排的IgG重链可变区序列;初步证实可变区序列不仅在V_H3-D-J_H-C_γ组合方式上具有极大的多样性,而且在序列上也具有明显的差异性;基于可变区测序对IgG抗体或B细胞进行分类是可行的,但需大大提高挑取克隆的数量或改用新一代大规模测序技术。     

抗CD3单克隆抗体重、轻链可变区基因的克隆及序列分析

目的：抗CD3单克隆抗体（WuT3)可变区基因克隆及序列分析。方法；采用RT-PCR技术，从WuT3杂交瘤细胞总RNA中扩增VH，VL片段，经酶切后通过链接反应构建重组克隆载体，测序鉴定。结果：通过国际联机检索发现VH，VL基因与Ig同源，分别符合小鼠IgVH,Vк基因特征。VH基因属于鼠重链VH第ⅡB亚类，全长363bp，可编码121个氨基酸；VL基因属于鼠к轻链第Ⅲ亚类，全长330bp，可编码110个氨基酸，结论：成功获得WuT3单抗的重，轻链可变区基因。     

抗人CD154单抗轻重链可变区基因克隆与序列分析

目的:克隆抗人CD154抗体轻重链可变区基因,并分析其核苷酸序列,为基因工程抗体的构建奠定基础。方法:从分泌能抑制免疫反应的抗人CD154单克隆抗体杂交瘤细胞株 7E8中提取总RNA,合成cDNA第一链后,经PCR扩增获得抗人CD154单抗轻链可变区 (VL)和重链可变区 (VH)基因,分别克隆入pUC18载体,并进行序列分析。结果:①抗体的轻链可变区基因全长为 341bp,编码113个氨基酸,归属于Ig的Vκ2基因,氨基酸序列分析结果显示轻链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3位和第 93位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸;②抗体的重链可变区基因全长为 354bp,编码118个氨基酸,归属于小鼠IgVH基因,D、J区基因分别属于DSP2.9和JH2,氨基酸序列分析结果显示,重链可变区含有明确的 4个骨架区和 3个抗原决定簇互补区,在第 2 3和第 97位氨基酸为半胱氨酸,是与抗体二硫键形成有关的两个特征性氨基酸。结论:经核苷酸序列分析证明所克隆的基因分别为抗体的轻、重链可变区基因.     

人噬菌体抗体库中异常重组子的分析

目的 阐明在噬菌体抗体库技术中，选择适当限制性内切酶酶切位点的重要性，并介绍人抗体可变区胚系基因的限制酶谱。方法 从正常人外周血提取淋巴细胞总RNA，用RT－PCR扩增IgM和IgG1的Fd片段及κ链基因，重组型载体p3MH中构建噬菌体抗体库。以限制性内切酶消化及电泳分析所获重组克隆；用PCGENE软件分析人抗体可变区胚系基因的限制性内切酶谱。结果 在构建抗体库的过程中，发现高频率的异常重组子克隆。经序列分析证实，在人VH基因片段中，存在用于克隆轻链的Sac I 位点。对人全部功能性可变区胚系基因进行限制性内切酶谱分析，发现人VH等Ⅳ家族的11个成员均含有SacI位点，其它限制酶切位点在抗体可变区胚系基因中具有不同的出现率。结论 构建抗体库时，用于重组可变区基因的酶切位点，对库的构建具有重要的影响，因此，应对表达载体所用的限制性内切酶进行精心选择。现在比较广泛使用的pCOMB系统载体不利于良好性能抗体库的构建。     

骨髓瘤细胞免疫球蛋白重链基因可变区的研究

为分析多发性骨髓瘤 (multiplemyeloma,MM )细胞对免疫球蛋白重链基因可变区 (VH)基因家族的取用 ;根据VH 基因突变特点 ,揭示MM细胞的起源。以重链基因可变区 (VH1 VH6 )基因家族特异性引物 ,用PCR法扩增骨髓瘤细胞系CZ 1细胞和 98例MM患者外周血单个核细胞VH 基因片段 ,纯化后的PCR产物和pMD18 T载体连接并转化JM10 9细菌 ,经克隆鉴定后 ,目的DNA片段用末端双脱氧法测定DNA序列 ,和其对应的胚系基因序列比较。结果表明MM细胞对各VH 基因家族的取用顺序为VH3>VH1>VH4 >VH2 >VH5 >VH6 ;MM细胞VH 基因互补决定区 (CDR )氨基酸替换性突变 (R突变 ) /氨基酸静寂性突变 (S突变 )等于 9 6 7,而骨架区 (FR )R/S等于 0 87,而且随着疾病的进展 ,IgVH 基因并不发生进一步的突变。结论是MM前体细胞在进行VDJ基因重排时 ,对VH 基因家族的取用和基因家族相对大小有关 ;MM细胞可能起源于已经发生抗原选择和体细胞突变的B记忆细胞或前浆细胞。     

人源性抗汉坦病毒抗体VH基因噬菌体表面展示文库的构建与筛选

目的 构建人抗汉坦病毒（HV）抗体重链可变区（VH）基因噬菌体表面展示文库,筛选人源性抗HV单克隆抗体（mAb）。方法 从HV吸附筛选的特异性人外周血B淋巴细胞中,用RT－PCR扩增VH基因,与噬菌粒pHEN1连接,构建VH基因噬菌体表面展示文库。经3轮吸附－洗脱－扩增的亲和筛选后,用ELISA鉴定抗HV噬菌体抗体的活性。结果 HV吸附筛选的特异性人外周血B淋巴细胞中,成功扩增出VH基因,并构建了VH基因噬菌体表面展示文库。经3轮亲和筛选,获得24个具有与HV结合活性的重组噬菌粒克隆。对其中一个克隆的VH基因（VH－D10）的序列分析表明,该基因的全长为363bp,编码121个氨基酸,与EMBL和GenBank中所有已知免疫球蛋白（Ig）基因进行同源性比较,其同源序列均人为IgVH原因。结论 成功地构建了人抗HV抗体VH基因噬菌体表面展示文库,并筛选到有HV结合活性的重组噬菌体克隆,为制备人源性抗HVmAb奠定了基础。     

AMPLIFICATION AND CLONING OF THE VARIABLE GENES OF MOUSE MONOCLONAL ANTIBOBY TO HUMAN C1q BY IN-CELL PCR AND SEQUENCE ANALYSIS

免疫球蛋白可变区信息包含了Ｂ淋巴细胞克隆产生的分子机制，对了解抗原、抗体间相互作用及在自身免疫中的作用机制具有重要意义。本研究应用两对通用引物，采用细胞内ＰＣＲ，从分泌高亲和力抗人Ｃ１ｑ单抗杂交瘤细胞扩增其轻、重链可变区基因。即杂交瘤细胞以４％多聚甲醛固定，经ＮＰ４０处理增加膜通透性后，直接作细胞内反转录合成ｃＤＮＡ，继以ｃＤＮＡ为模板作细胞内ＰＣＲ，从而无需提取细胞ＲＮＡ或ＤＮＡ便扩增获得轻、重链Ｖ基因。同时，通过建立快速、高效、高分辨率毛细管电泳技术检测ＰＣＲ扩增产物，达到ＰＣＲ扩增及检测的自动化。扩增的轻、重链可变区基因分别克隆进ｐＧＥＭ－１１Ｚｆ（－）并作序列测定。计算机序列分析及同源性检索证实所克隆基因片段归属鼠免疫球蛋白家族。按Ｋａｂａｔ分类标准，抗－Ｃ１ｑ轻链Ｖ基因（Ｖκ）属鼠Ｉｇ第Ｖ组，而其重链Ｖ基因（ＶＨ）属ⅡＢ组。令人感兴趣的是，抗－Ｃ１ｑＶκ与自身抗体的Ｖκ高度同源，而抗－Ｃ１ｑＶＨ主要与胚系基因及某些自身抗体显著同源（达７５％以上）。由胚系基因编码的不同特异性抗体往往具有与自身抗原结合特性，自身抗体亦多由胚系基因编码。因此，本株高亲和力抗－Ｃ１ｑＩｇＧ与胚系基因及自身抗体Ｖ基因密切相     

U251细胞血清饥饿特异抗原的ScFv噬菌体抗体库构建

目的：构建一个U251细胞血清饥饿特异抗原ScFv噬菌体抗体库。方法：将U251细胞进行48小时的血清饥饿培养,将其细胞裂解液免疫4周龄的BALB/c小鼠,提取被免疫小鼠脾脏细胞总RNA,反转录成cDNA,利用RT-PCR扩增免疫球蛋白IgG的重链可变区（VH）和轻链可变区（VL）基因,用一个柔性片段（Linker）连接VL和VH基因片段,将连接产物重组到pCANTAB-5E载体,转入TG1菌株中,随后加入辅助噬菌体（Help phage）M13K07超感染,构建单链抗体片段（Single chain fragment of variation,ScFv）噬菌体抗体库。结果：经过富集筛选后,库容量达到3×10^6cfu/L,随机挑取8个克隆进行ELISA检测,获得了1个阳性克隆。结论：成功构建了一个具有一定库容的U251细胞血清饥饿特异抗原的单链抗噬菌体抗体库,为筛选血清应答蛋白抗体、进一步克隆血清应答蛋白的基因奠定基础。     

从兔外周血直接克隆B细胞κ轻链的可变区

目的 从兔外周血总RNA中直接扩增出B细胞κ轻链的可变区序列.方法 首先从IMGT/GENE-DB数据库中获取大耳白兔Ig胚系基因Vκ(IGKV)、Jκ(IGKJ)和Cκ(IGKC)的cDNA序列、设计引物,然后以非免疫兔外周血总RNA为模板进行RT-PCR扩增,回收产物并克隆到T载体,随机挑选单克隆测序,最终将序列提交到IMGT/V-QUEST,分析出每个克隆的Vκ-Jκ组合,并利用BioEdit的本地Blast程序比较出Cκ的基因型.结果 共设计出4对引物,其中引物对RK.C1[4-5-9]无产物,引物对RK.C1[1-2-7]、RK.C1[3-6-8]和RK.C2[1-2-3-4]有产物,分别回收、克隆到T载体后各挑取20个克隆,共获得51个克隆的插入子序列.没有任何2个克隆的插入子序列完全相同,每个克隆均包含完整的兔Vκ、Jκ及Cκ的5'端序列,其中Vκ-Jκ-IGKC1组合的克隆33个,Vκ-Jκ-IGKC2组合的克隆18个;68个Vκ胚系基因中有22个出现,其中频率最高的是IGKV1S10*01(12次),其次是IGKV1S36*01、IGKV1S37*01和IGKV1S4*01(各5次),其余均为3次以下;8个Jκ胚系基因中只有1个出现,为IGKJ1-2*01;2个Cκ基因的等位基因分别为IGKC1*01和IGKC2*03.33个Vκ-[IGKJ1-2*01]-[IGKC1*01]克隆中有17种不同的组合方式,其中频率最高的为[IGKV1S10*01]-[IGKJ1-2*01]-[IGKC1*01](7次).18个Vκ-[IGKJ1-2*01]-[IGKC2*03]克隆中有11种不同的组合方式,频率最高的为[IGKV1S10*01]-[IGKJ1-2*01]-[IGKC2*03](5次).Vκ-Jκ-Cκ组合方式相同的克隆,序列仍具有明显的差异.结论 利用自行设计的兼并引物,成功并特异地从兔外周血总RNA中直接扩增出了κ轻链的可变区,且产物具有良好的多样性,但所有Vκ-Jκ-Cκ组合中只出现了1种Jκ基因.     

鼠抗hTNF—α单克隆抗体重链基因片段的克隆和cDNA序列测定

采用反转录ＰＣＲ方法，以一对锚ＰＣＲ引物和一对针对鼠ＩｇＶＨ区基因的引物，从分泌鼠抗ｈＴＮＦ－α单克隆抗体的Ｅ６杂交瘤细胞株中，分别扩增和克隆了自５′非翻译区至Ｃγ１近３′端的重链基因片段和重链可变区基因片段，并测定了其核苷酸序列。计算机分析表明，系重排的鼠Ｉｇ重链基因。     

The analysis of VH and VL genes repertoires of Fab library built from peripheral B cells of human rabies virus vaccinated donors

A human combinatorial Fab antibody library was generated from immune repertoire based on peripheral B cells of ten rabies virus vaccinated donors. The analysis of random Fab fragments from the unselected library presented some bias of V gene usage towards IGHV-genes and IGLV-gen families. The screening of the Fab library on rabies virus allowed specific human Fab antibody fragments characterized for their gene encoding sequences, binding and specificities to RV. Genetic analysis of selected Fabs indicated that the IGHV and IGLV differ from the germ-line sequence. At the level of nucleotide sequences, the IGHV and IGLV domains were found to share 74–92% and 90–96% homology with sequences encoded by the corresponding human germ-line genes respectively. IGHV domains are characterized most frequently by IGHV3 genes, and large proportions of the anti-RV heavy chain IGHV domains are obtained following a VDJ recombination process that uses IGHD3, IGHD2, IGHD1 and IGHD6 genes. IGHJ3 and IGHJ4 genes are predominantly used in RV-Fab. The IGLV domains are dominated by IGKV1, IGLV1 and IGLV3 genes. Numerous somatic hypermutations in the RV-specific IGHV are detected, but only limited amino acid replacement in most of the RV-specific IGLV particularly in those encoded by J proximal IGLV or IGKV genes are found. Furthermore, IGHV3–IGKV1, IGHV3–IGVL1, and IGHV3–IGLV3 germ-line family pairings are preferentially enriched after the screening on rabies virus.     

Natural human antibodies to pneumococcus have distinctive molecular characteristics and protect against pneumococcal disease

The molecular and functional characteristics of natural antibody from the preimmune repertoire have not been explored in detail in man. We describe seven human IgM monoclonal antibodies selected on the basis of pneumococcal polysaccharide binding that share both molecular and functional characteristics with natural antibody, suggesting a common B cell lineage origin. Unlike class-switched antibodies, which are serotype-specific, the antibodies were polyreactive and bound all pneumococcal polysaccharide capsular serotypes tested. Some bound endogenous antigens, including blood group antigens and intermediate filament proteins. All the antibodies used unmutated heavy chain V (IGHV) that are expressed at an increased frequency in the elderly and in the preimmune repertoire. The CDR3 was characterized by long length (mean aa 18.4 (+/-4.2) and selective use of IGHD6 (P < 0.001) and IGHJ6 (P < 0.01) family genes. The clones expressing IGHV1-69 and IGHV 3-21 provided significant passive protection against invasive pneumococcal disease in vivo.     

The Teleostei immunoglobulin heavy IGH genes

'Teleostei Immunoglobulin Heavy IGH Genes', the eleventh report of the 'IMGT Locus in Focus' section, comprises four tables: (1) 'Teleostei IGHV genes'; (2) 'Teleostei germline IGHJ genes'; (3) 'Teleostei IGHC genes and alleles'; (4) 'FR-IMGT and CDR-IMGT length of the Teleostei IGHV genes'. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr: 8104) created in 1989 by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.     

Rearrangement of only one human IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses in transgenic mice

In recent years, mice carrying human IG transgenes are being generated for the production of human monoclonal antibodies as an alternative approach to the conventional use of mouse or chimeric-humanized antibodies. Theoretically, the size of the repertoire of human antibodies that these mice could produce would be critically dependent on the number of human V genes introduced in the transgene. This could be the case for BABkappa and BABkappa,lambda transgenic mice, which carry several genes from the human IGK (BABkappa), and IGK and IGL (BABkappa,lambda) loci, but only five human IGHV genes and the entire IGHD-IGHJ cluster linked to two human IGHC (IGHM-IGHD) genes. We analyzed the expressed human IG genes in 30 IgM-secreting hybridomas generated from transgenic mice immunized either with soluble proteins (human IgM coupled to KLH) or with cells (human PBMC, tumour cell lines or rat cells transfected with human CD69). The results show that all hybridoma cells analyzed rearranged exclusively the IGHV1-2 gene, in contrast with naive spleen B cells that used three out of the five IGHV genes present in the transgene. The configuration of the rearranged CDR3 region revealed a much higher heterogeneity in the heavy chains. A variety of IGHJ and IGHD genes were used in hybridomas, and somatic mutations were also seen in some hybrids. Regarding the rearranged light chains genes, it was a much higher variety in the use of V and J genes in both, kappa and lambda chains, than in the heavy chain, and also in the level of mutation. The results indicate that only one IGHV gene is sufficient to generate a wide repertoire of antigen specific antibody responses. Thus, efforts aimed at the generation of new transgenic mice should focus more on the integrity of the D/J region and on the DNA regions regulating somatic hypermutation, rather than on the number of V genes present in the transgene.     

Th17和Treg细胞以及其相关细胞因子在慢性丙型肝炎以及丙型肝炎肝硬化病人外周血中的变化及意义

目的：〖HTSS〗研究Th17和Treg细胞以及其相关细胞因子在慢性丙型肝炎以及丙型肝炎肝硬化病人外周血中的变化及意义,以探讨其在慢性丙型肝炎及丙型肝炎肝硬化发病中的作用。 〖HTH〗方法：〖HTSS〗用流式细胞和ELSIA技术检测慢性丙型肝炎及丙型肝炎肝硬化病人外周血Th17和Treg细胞表达率以及IL 6、IL 10、TGF β、IL 17血清学水平,分析上述指标在健康对照组和慢性丙型肝炎组以及肝硬化组之间的差异。 〖HTH〗结果：〖HTSS〗慢性丙型肝炎病人的Th17细胞表达率(133%±030%)以及IL 6［(810±242）ng/L］、IL 17［(1670±473）ng/L］血清水平明显高于健康对照组Th17 细胞表达率(114%±019%)以及IL 6［(670±172）ng/L］、IL 17［(1229±188）ng/L］血清水平,P＜005;丙型肝炎肝硬化组和慢性丙型肝炎组病人外周血的Treg细胞表达率(621%±076%,589%±085%)明显高于健康对照组的Treg细胞表达率（551%±059%）,P<005;丙型肝炎肝硬化组病人Th17/Treg的比值（019±002）明显低于慢性丙型肝炎组（022±003）和健康对照组（021±003）,P<005;丙型肝炎肝硬化组外周血IL 10水平［（1621±376）ng/L］和TGF β水平［（515±083）ng/L］显著高于慢性丙型肝炎组［（1436±278）ng/L;（447±087）ng/L］和健康对照组［（1401±301）ng/L;（443±098）ng/L］,P<005。 〖HTH〗结论：〖HTSS〗Th17和Treg细胞以及其相关细胞因子参与了丙型肝炎慢性化和肝硬化的进程,Th17和Treg细胞以及其相关细胞因子在丙型肝炎慢性化和肝硬化的治疗和预防方面可能具有重要的意义。     

Production of recombinant Ig molecules from antigen-selected single B cells and restricted usage of Ig-gene segments by anti-D antibodies

The Ig-genes of the heavy chains in anti-D-specific hybridomas and Fab/scFv-fragments selected from phage-display libraries are restricted to a group of closely related genes (IGHV3s genes). We analyzed the Ig-gene repertoire in anti-D-specific B cells of two hyperimmunized donors using a completely different method. Single B cells were cultured for 10 days in an EL4.B5 culture system. mRNA from anti-D-producing B cells was reverse transcribed into cDNA. Heavy- and light-chain gene rearrangements were amplified by PCR reactions, sequenced and cloned into a pNUT-vector system, thereby allowing the production of complete IgG and IgM. Eleven anti-D-specific B-cell clones were isolated and analyzed. Eight of these clones (including IgM-producing clones) had IGHV3s genes. We demonstrated that functional anti-D-specific IgM (4 clones) and IgG (2 clones) was produced. Using a new method, we analyzed the IGHV gene repertoire of anti-D-specific B cells of hyperimmunized donors and showed that it is indeed restricted. Moreover, we found a high frequency (1:100 and 1:500) of anti-D-specific B cells in the peripheral B cells of hyperimmunized donors. We suggest that this approach could be applied for the selection of human mAbs from immunized donors and for the analysis of Ig-gene repertoires at the single-B cell level.     

Extensive restrictions in the VH sequence usage of the human antibody response against the Rhesus D antigen

Anti-Rhesus D immunoglobulin purified from human sera is used as a prophylactic reagent in Rhesus D negative women at risk of alloimmunization during pregnancy. We are currently developing a Rhesus D antigen-specific recombinant polyclonal antibody drug lead for replacing the existing blood derived-products. By analyzing the RhD-specific antibody VH repertoires from eight alloimmunized women we found, in agreement with previous studies, a strong preference for the VH 3-33 "superspecies" gene segments which encompasses the IGHV3-30-3*01, IGHV3-30*18, and IGHV3-33*01 VH alleles. Even more extensive genetic restriction was observed among five donors, which produced antibodies of identical V-D-J usage and CDR3 loop length and joining regions of similar amino acid composition. In addition, we find a high degree of sequence relatedness to previously isolated anti-Rhesus D antibodies. Such close homology in VH domains indicates that significant structural restrictions are operating in the selection of antibodies recognizing RhD as seen for T cell receptors. Moreover, some VH domains were isolated in their germline configuration indicating that anti-RhD antibodies of relatively high affinity are present in the na?ve antibody repertoire of Rhesus negative individuals which offers an explanation for the strong and clinically significant immunogenicity of the Rhesus D.