扩增抗体重链可变区的一个高效方法

从杂交瘤细胞或脾细胞中扩增抗体可变区，是构建单链抗体（Single-chain Fv fragment，ScFv）、克隆单克隆抗体和研究抗原与抗体相互作用的关键一步。而抗体可变区（Variable regions，Fv）高度变异，扩增相对困难，至今仍是一个难点，有待解决。我们在构建ScFv过程中，发现2株杂交瘤细胞用普通方法难于扩增出抗体重链可变区（Heavy—chain variable region，VH）抗体。于是提出了一种多序列比对和简并引物设计算法，并加以实现，设计出通用的鼠源性抗体VH引物；应用上述引物，采用反向多聚酶链反应（Reversepopymerase chain reaction，reversePCR）方法——3’RACE和5’RACE法，准确地扩增出2株杂交瘤细胞的VH基因。该算法很好地解决了引物特异性和最小化问题，具有简单、实现容易的特点，可用于基因家族中未知基因克隆和文库构建中的引物设计。与反向PCR方法结合，提高了难扩增的未知基因的成功率。     

Use of family specific leader region primers for PCR amplification of the human heavy chain variable region gene repertoire.

We have designed a set of six, non-degenerate oligonucleotide primers, corresponding to the 5' leader regions of each of the six human VH gene families. A general strategy for family specific polymerase chain reaction amplification is described using these primers and a conserved 3' primer corresponding to frame work 3, JH, or constant region. This strategy was used to isolate and sequence novel human germline VH genes belonging to the VH2 and VH4 families. Under certain conditions, chimeric VH sequences were created by a "jumping polymerase chain reaction", combining DNA segments from different germline genes, but this could be avoided by limiting the number of amplification cycles. PCR amplification with these family specific primers will facilitate studies of the repertoire of germline VH genes as well as studies on VH gene usage in normal and aberrant (B cell malignancies, autoimmune diseases, etc.) B cell populations.     

Repertoire of human antibodies against the polysaccharide capsule of Streptococcus pneumoniae serotype 6B

We examined the repertoire of antibodies to Streptococcus pneumoniae 6B capsular polysaccharide induced with the conventional polysaccharide vaccine in adults at the molecular level two ways. In the first, we purified from the sera of seven vaccinees antipneumococcal antibodies and determined their amino acid sequences. Their VH regions are mainly the products of VH3 family genes (candidate genes, 3-23, 3-07, 3-66, and 3-74), but the product of a VH1 family gene (candidate gene, 1-03) is occasionally used. All seven individuals have small amounts of polyclonal kappa+ antibodies (Vkappa1 to Vkappa4 families), although kappa+ antibodies are occasionally dominated by antibodies formed with the product of the A27 Vkappa gene. In contrast, lambda+ anti-6B antibodies are dominated by the antibodies derived from one of 3 very similar Vlambda2 family genes (candidate genes, 2c, 2e, and 2a2) and Clambda1 gene product. The Vlambda2(+) antibodies express the 8.12 idiotype, which is expressed on anti-double-stranded-DNA antibodies. In one case, Vlambda is derived from a rarely expressed Vlambda gene, 10a. In the second approach, we studied a human hybridoma (Dob1) producing anti-6B antibody. Its VH region sequence is closely related to those of the 3-15 VH gene (88% nucleotide homology) and JH4 (92% homology). Its VL region is homologous to the 2a2 Vlambda2 gene (91%) and Jlambda1/Clambda1. Taken together, the V region of human anti-6B antibodies is commonly formed by a VH3 and a Vlambda2 family gene product.     

Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3' to 5' exonuclease activity

Degenerate primers were designed for PCR amplification of unknown mouse immunoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each subgroup of mouse Ig gene sequences [Kabat, E.A., Wu, T. T., Perry, H.H., Gottesman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Interest, 5th edn. US Department of Health and Human Services, Public Health Service, NIH.] was analyzed, and highly degenerate primers in the framework one (FR1) region were designed. A single highly degenerate FR1 primer sufficed for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3' to 5' exonuclease activity of DNA polymerase on the utilization of these degenerate primers. Using Taq polymerase, which lacks 3' to 5' exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hundred monoclonal hybridoma cell lines reactive against a phosphonamidate hapten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed that they are derived from multiple germline families (10 of the 17 VL families and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse (Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The universality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens.     

具有GPX活性的单克隆抗体可变区基因的克隆和序列分析

目的利用分泌具有GPX活性的单克隆抗体(mAb)的杂交瘤细胞3G5,克隆其mAb体的可变区基因。方法提取杂交瘤细胞的总RNA ,分离mRNA ,反转录合成cDNA。经PCR扩增VH 基因和VL 基因 ,将VH 基因和VL基因与载体 pGEM T连接后 ,进行酶切鉴定和序列分析。结果构建了2个分别含有VH 和VL 基因的重组质粒。序列分析表明 ,VH 和VL 分别属于mouscheavychainsubgroupIII和mouselightchainsubgroupV亚群 ,长度为372和324bp ,编码124和108个氨基酸 ,在高变区分别有4和2个丝氨酸。结论克隆的VH 和VL 基因符合功能性重排的鼠抗体可变区基因特征 ,为将来制备具有GPX活性的单链抗体提供了可靠的基因材料。     

Immunoglobulin variable regions usage by B-lymphocytes infiltrating a human breast medullary carcinoma

Breast medullary carcinoma are heavily infiltrated by B-lymphocytes and associated with a good prognosis despite their high histological grade. We investigated the Ig repertoire of B-lymphocytes infiltrating one such tumour. A single cell suspension was obtained from a tumor specimen by enzymatic digestion. VH, Vkappa, and Vlambda regions were amplified by RT-PCR using mixtures of primers optimized to maximize the diversity of the PCR products. They were then cloned and sequenced. Analysis of 9 VH, 5 Vkappa, and 10 Vlambda sequences using the Kabat database indicated that several VH and VL region subgroups (I, II and III) are expressed by B-lymphocytes infiltrating this tumor. The analysis of CDR3 regions also showed a variability, although some VH and VL clones exhibited identical or nearly identical sequences. Thus, the B-cell infiltration observed in this breast medullary carcinoma does not reflect a monoclonal proliferation and represents an oligoclonal or a polyclonal B-cell proliferation.     

抗人核内不均一核糖核蛋白A2/B1抗体可变区基因克隆、串联和表达

目的克隆抗人核内不均一核糖核蛋白A2/B1(heterogeneousnuclearribonucleoproteinA2/B1,HnRNPA2/B1)单克隆抗体的重链可变区(variableregionofheavychain,VH)和轻链可变区(variableregionoflightchain,VL)基因,构建抗HnRNPA2/B1单链抗体(singlechainFv,ScFv)基因,并在大肠杆菌表达。方法采用斑点ELISA、Western印迹和免疫组化检测抗HnRNPA2/B1单克隆抗体3E8的特异性,并通过逆转录-聚合酶链反应、重叠延伸PCR来克隆、串联VH和VL基因,构建抗HnRNPA2/B1ScFv基因pET28(a )表达载体,在大肠杆菌BL21中诱导表达,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳、竞争抑制ELISA检测表达产物。结果3E8抗体能特异性地与HnRNPA2/B1合成肽以及3株人肺癌细胞内HnRNPA2/B1结合;克隆的VH基因为345bp,VL基因为309bp,符合小鼠抗体可变区特性;抗HnRNPA2/B1ScFv蛋白以包涵体形式表达,分子量约28000,并具有与抗原结合活性。结论成功构建了抗HnRNPA2/B1ScFv基因克隆,并在大肠杆菌中获得了功能性表达,为阐明HnRNPA2/B1与肺癌相关性奠定了实验基础。     

Preferential VH/V lambda pairings occur in the available B cell repertoire of adult BALB/c mice.

To gain insights into the composition of the B cell repertoire, we have investigated VH gene family expression associated with individual light chains. For this purpose, we have examined the use of 12 VH gene families in a large collection of hybridomas expressing one of the four lambda light chains [lambda 1 (V1J1), lambda 2 (V2J2 and V x J2) and lambda 3 (V1J3)]. Our results show that the distribution of the VH families is very different from one lambda subtype to another. This suggests that a few substitutions between VL regions are sufficient to generate very different associated repertoires by strong selection mechanisms. Moreover, we assume that the global VH expression pattern is not random but rather composed of many preferential VH/VL associations.     

The antibody site in Atlantic salmon; phage display and modeling of scFv with anti-hapten binding ability.

Cloning and sequencing the cDNA of around 50 VH (VDJ) and 15 VL genes in Atlantic salmon demonstrated nine VH families (above 80% identity within each family) and one dominating but relatively diverse VL family in this species. The highest variability of the VH was seen in the CDR3, but CDR2 also expressed a modest variability. The 'whole' antibody repertoire was expressed as single chain Fv (scFv) in a phage display library by combining 12 VH and two VL specific primers (FR1/microl and FR1/CL, respectively). The PCR products (VH and VL) were ligated (with a G-rich spacer) into the lambda Surf-Zap (Stratagene) vector and expressed as a surface fusion protein on the M13 phage. Anti-TNP and anti-FITC specific scFv clones were isolated by panning using hapten-coated magnetic beads and the coding DNA sequenced. The specificities of the anti-TNP and anti-FITC clones were similar to mouse monoclonal antibodies. 3D-models of the active sites (CDRs) of the anti-TNP and anti-FITC clones suggest hapten-interacting structures of the salmon antibody site similar to mammalian antibodies.     

Heterogeneity of cross-reactive idiotypes. Serological and structural analysis of monoclonal anti-p-azobenzene-arsonate antibodies expressing major and minor idiotypes

Hybridoma-derived monoclonal anti-p-azobenzene-arsonate (ABA) antibodies were obtained from fusions of ABA-KLH primed A/J spleen cells with three different myeloma cell lines. Of the 156 antibody secreting hybridomas 24% carried the cross-reactive idiotype (CRI), which is known to be shared by 20-70% of anti-ABA serum antibodies in A/J mice. The isotypes, SDS-PAGE patterns and the partial amino acid sequences of the V-regions of one CRI negative and six CRI positive hybridoma proteins were determined. These antibodies were IgGl, kappa and IgG2b, kappa. Some idiotype carrying monoclonal antibodies appeared to be serologically identical. Although the partial VH amino acid sequences of these monoclonal antibodies showed great homology with each other and with serum antibody, several sequence variations in framework residues as well as in the first and second complementarily determining regions (CDRs) were found. The cross-reactive idiotype of the anti-ABA antibodies, therefore, exhibits structural microheterogeneity, i.e. it consists of a family of non-identical but closely related molecules as previously reported (Alkan et al., 1980; Estess et al., 1980; Marshak-Rothstein et al. 1980). Here the N-terminal sequence of the VH regions from 14 CRI+ and 8 CRI- antibodies as well as the VL regions from 11 CRI+ and 8 CRI- monoclonal antibodies are compared. Analysis of the available data demonstrated that there are pairs of hybridoma proteins (both CRI+ and CRI-) which have identical sequences for VH or VL. This suggests that there exist a minimum of 4 germ line genes coding for CRI+ VH, CRI+ VL, CRI- VH and CRI- VL respectively. In addition, CRI+ VL has always been found in association with a CRI+ VH.     

中国人群慢性淋巴细胞白血病免疫球蛋白重链可变区基因组成及突变分析

目的 研究中国慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL)患者免疫球蛋白重链可变区(immunoglobulin variable heavy chain region,IGVH)基因各个片段的组成以及突变情况.方法 应用多重PCR技术扩增64例CLL患者的IGVH基因片段,纯化PCR扩增产物后直接测序,应用IMGT/V-QUEST及IGBlast数据库分析,明确VH基因有无突变和突变位置,以及VH、DH,JH家族各个成员组成情况.结果 64例CLL患者中,VH3家族31例(48%)、VH4家族26例(41%)、VH1家族4例(6%)、VH2家族2例(3%)、VH7家族1例(2%).44例患者发生体细胞突变,占CLL患者的69%;20例无突变,占CLL患者的31%,其中6例(9%)VH基因的同源性为100%.VH4家族中有9例无突变(9/26,35%);VH3家族中8例无突变(8/31,26%);VH1家族中1例无突变(1/4,25%).在所检测的CLL标本中,VH4-34是最常见的VH基因片段,检测出8例(13%),其中无突变3例;其次为VH4-59,检测出7例(11%),无突变者3例.DH基因中DH3家族最常见,有25例(39%);其中11例(11/20,55%)出现在无突变组中.无突变组中最常见的DH基因片段为DH3-10和DH3-22,各为4例(4/20,20%).JH基因中JH6家族最常见,检测出23例(36%),其中9例出现在无突变组中(9/20,45%).结论 中国CLL患者IGVH基因家族表达比例和突变情况与西方国家存在显著差异,推测可能与不同的种族和环境中的抗原选择有关,其预后意义有待进一步探讨.     

抗血小板膜糖蛋白单抗SZ—21嵌合Fab片段基因的构建和表达研究

目的 为降低抗血小板膜糖蛋白单克隆抗体SZ－21的免疫原性,克隆了SZ－21可变区基因,构建并表达人－鼠嵌合Fab基因工程抗体。方法 利用基因克隆技术,从杂交瘤细胞系SZ－21克隆出轻、重链可变区基因,然后亚克隆到质粒pSW1中,构建成人－鼠嵌合Fab片段基因质粒pSZ-21,在大肠杆菌中表达。结果 pSZ－21基因构建正确,在大肠杆菌中表达出可溶性抗体片段,表达产物具有与血小板结合的活性。结论 成功地克隆了SZ－21可变区基因,并表达了可溶性人－鼠嵌合Fab基因工程抗体。     

Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes

In recent work, the polymerase chain reaction (PCR) has been used to amplify rearranged mouse and human immunoglobulin heavy and kappa light chain variable (V) genes. Here we have optimized the design of the PCR primers for human V genes and used them to amplify cDNA from human peripheral blood lymphocytes. Cloning and sequencing revealed a diverse repertoire of V genes, and the presence of members of each human V gene family. After alignment of the sequences, we identified a region conserved within V gene families, but differing between families, and used this to design family-specific oligonucleotide probes.     

Variable region genes of human monoclonal autoantibodies to histones H2A and H2B from a systemic lupus erythematosus patient

An antibody phage library obtained from peripheral blood lymphocytes of a systemic lupus erythematosus (SLE) patient was used to isolate four monoclonal autoantibodies against histones H2A and H2B. Analysis of the variable region sequences revealed that the anti-histone monoclonal antibodies were not clonally related; they used VH genes from three different VH gene families (VH3, VH4, and VH5) and distant members of the Vkappa group (L25, L6, A27, and O8) in conjunction with different D and J gene segments. These observations suggest that certain gene families or segments are not critical in producing anti-histone autoantibodies in SLE. Most of the utilized VH and Vkappa sequences were highly mutated and the complementarity-determining regions (CDRs) varied greatly in length. The VH region of the antibody SLEhis18 had an isoelectric point of 6.1, and 29% of the mutations were changes to acidic amino acid residues. The second CDR (CDR2) of SLEhis18 VH contained one basic and three acidic residues. Acidic residues were observed in the CDR3 regions of VH, but not VL, in all isolated clones; this is unusual, as most autoantibodies are comprised predominantly of non-acidic residues. This is the first report of a systematic sequence analysis of human anti-histone monoclonal antibodies. Our results suggest that certain V genes are not important for autoreactive specificity to histones in SLE; instead, other mechanisms such as an existence of acidic residues and somatic mutations in CDRs are required for specific binding to histones, which might play a role as a stimulatory autoantigen for the activation of autoantibody-producing B-cells and the selection of high affinity antibody.     

Characterization of human hybridoma clones isolated from hemophilia patients with specificity for different domains of coagulating factor VIII

Hemophilia A patients treated with human coagulating factor VIII (FVIII) may develop inhibitory antibodies (inhibitors). Characterization of the inhibitors at the clonal level may help exploring new therapeutic strategies. We have generated lymphoblastoid cell lines (LCLs) producing anti-FVIII antibodies from peripheral blood lymphocytes of hemophilia A patients with high inhibitor titers. We fused the anti-FVIII-positive LCLs with a heteromyeloma, to produce FVIII specific hybridomas. We determined the specificity, isotype, idiotypic and immunoglobulin (Ig) variable region heavy (VH) chain gene family profiles of the secreted antibodies (Ab) by ELISA, immunoblotting and RT-PCR. We established eight hybridomas which produced high titers of anti-FVIII Ab. All hybridomas secreted IgM Ab, associated with either kappa(5/8) or lambda(3/8) light chain. Analysis of the expressed VH genes by RT-PCR revealed that the hybridomas utilized only the VH1 (63%) or the VH3 (37%) gene families. Among the cross-reactive idiotypes (CRIs) we tested, only the VH1 and VK3b-associated CRIs were expressed by 3 hybridomas. Immunoblotting of thrombin-digested FVIII demonstrated distinct patterns of reactivity of the monoclonal Ab (MAb) secreted by the hybridomas, which recognized either the A2 domain of the Fvm heavy chain, or the light chain, or both. Our findings suggest that: a) the isotype of the anti-FVIII Ab secreted by LCLs and hybridoma clones (IgM) differs from that of anti-FVIII Ab in vivo, which are predominantly IgG4: this suggests a negative selection of the isotype-switched FVIII-specific B-cells in the periphery of these patients; b) the anti-FVIII Ab have a biased representation of the VH1 gene family, and c) somatic mutations in the VH genes coding for FVIII specificity occur in the anti-FVIII Ab response, as evidenced by lack of expression of the VH-associated CRI.     

Molecular analysis of cross-reactive anti-myosin/anti-streptococcal mouse monoclonal antibodies

Nucleotide sequences of VH- and VL-genes of anti-myosin/anti-streptococcal monoclonal antibodies (mAbs) were analyzed and compared with their highly detailed antigen binding reactivities. Antigen-specificities of the cross-reactive mAbs included myosin, streptococcal M-protein, actin, keratin, N-acetyl-beta-D-glucosamine, vimentin, DNA, tropomyosin, troponin, and laminin as previously described. After nucleotide sequence analysis, homology indicated that some of the V gene sequences aligned with antibodies recognizing gangliosides and blood group antigens glycophorin M and N. Therefore, mAb reactivity with gangliosides and glycophorin M and N was identified. The cross-reactive mAbs utilized a heterogeneous group of germline V-heavy genes comprised of nine J558-, four 7183- and two Q52-family VH-genes. Germline V-light genes utilized by the mAbs included six Vkappa4/5-, three Vkappa8-, two Vkappa10-, three Vkappa19- and one Vkappa23-family VL-genes. No preferential VH/VL-chains correlated with any of the 12 different antigen reactivities, even for mAbs with nearly identical cross-reactivities. However, we did find that the cross-reactive mAb germline genes within a V gene family shared more homology among themselves than with other germline genes within their V gene families, suggesting convergent mutation. Cross-reactive mAbs with the highest relative avidity for myosin were found in the VH7183 family which contained two cytotoxic mAbs. Antibodies with V gene sequences most homologous to those of our cross-reactive anti-myosin/anti-streptococcal mAbs had specificities for laminin, DNA, carbohydrates, or blood group antigens and were reported to cause autoimmune disease in mice.     

Autoimmune VH gene family: PCR-generated murine germline VH10 genes.

A relatively large number of variable region genes (V) contribute, via gene rearrangements with smaller numbers of additional gene elements (D and J), to generate diversity in the immune response. While some VH gene families are thought to contain 100- 1000 members, the VH10 family has only two known functioning members with 99% sequence homology. Both members (monoclonal antibodies) are capable of binding DNA, and since they were derived from inbred mice afflicted with the lupus syndrome they are considered autoimmune antibodies. Relative uniqueness of the VH10 primary nucleotide sequence presents a model system with which to examine unrearranged VH genes and attempt to identify germline genes eventually expressed as autoantibodies. PCR amplified germline sequences of the VH10 family are highly conserved, with few base substitutions evenly distributed between both framework and CDR regions. It was determined that the PCR amplified germline sequences are highly similar to the DNA sequences of the two monoclonal VH10 antibodies, and a non-functional psuedo-germline gene was found that is identical to a non-functional cDNA derived from a hybridoma cell line. These findings indicate that the use of unique CDR DNA sequences for the identification and amplification of specific germline V genes via PCR can yield vital information that may answer fundamental questions about the origins of autoimmune anti-DNA antibodies in afflicted individuals. The nature of the germline gene populations and the possible microheterogeniety of these genes may prove to be important in understanding the role of autoimmune antibodies in normal and diseased individuals.