The immunoglobulin light chain locus of the turkey, Meleagris gallopavo

To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.     

Monoclonal antibodies against sheep immunoglobulin light chain, IgM and IgA

Monoclonal antibodies specific for sheep immunoglobulin light chain, IgM and IgA were produced by conventional cell fusion technology. Purified light chain and IgM were used to verify the specificity of anti-light chain and anti-IgM hybridoma supernatants using passive haemagglutination assays, radioimmunoassays and immunoelectrophoresis. In the absence of pure IgA, verification of monoclonal anti-IgA was, based on paired staining of intestinal lymph smears and comparing the percentage of cells stained with hybridoma supernatant with the percentage of cells stained with polyclonal anti-alpha serum.     

Developmental progression of immunoglobulin heavy chain diversity in sheep

In order to assess the respective impacts of combinatorial rearrangement, junctional diversification, somatic hypermutation and gene conversion in the generation of immunoglobulin heavy chain variable regions diversity, the sequences of 42 variable regions from late fetal, newborn and young sheep were determined and compared to those of adult animals. At earlier stages of development, the use of germline diversity segments appears restricted, junctional variability is already established, and somatic hypermutations are scarce. The sequence diversity in adults is much higher, which we suggest results from a higher hymermutation activity and possibly from the use of a variety of diversity segments. Altogether, this pattern is very reminiscent of the situation observed in cattle, except for the length of the third complementarity determining regions (CDR3) which are shorter in sheep than in bovine. Unlike the chicken and rabbit systems, it seems that new rearrangements continue to occur in sheep for at least several months after birth.     

Characterization of the bovine immunoglobulin lambda light chain constant IGLC genes

To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals.     

Generation and characterisation of murine monoclonal antibodies specific for cervine immunoglobulin light chain, IgM and IgG.

Monoclonal antibodies (mAb) which react with cervine immunoglobulin (Ig) light chain, IgM and IgG were produced using conventional cell fusion technology. Hybridoma supernatants were initially screened for specificity against cervine Ig using an enzyme-linked immunosorbent assay (ELISA). The specificity of supernatants against size-fractionated cervine Ig was further determined. Supernatants were characterised using western blotting and autoradiographic techniques. The mAb OU1G, OU2G and OU3G were specific for cervine gamma-chain of IgG, whereas OU1L was specific for light chain of Ig. A further mAb (OU1M) bound IgM and not IgG. These mAb were found to have varying cross-reactivity against Ig from other species.     

Somatic hypermutation leads to diversification of the heavy chain immunoglobulin repertoire in cattle

The availability of unique variable (VH), diversity (D), and joining (JH) gene segments in the vertebrate germline determines the extent to which a primary immunoglobulin (Ig) repertoire can be generated through combinatorial rearrangement. Although bovine D segments possess unusual properties, the diversity of the primary Ig heavy chain (IgH) repertoire in cattle is restricted by the dominance of a single family of germline VH genes of limited number and diversity. Cattle therefore must employ other diversification strategies in order to generate a functional IgH repertoire, the main candidates being gene conversion and somatic hypermutation. In considering these possibilities, we predicted that if somatic hypermutation was active during B lymphocyte development, the process would introduce nucleotide substitutions to the VDJ exon and also non-coding region lying downstream of the rearranged JH segment. In contrast, our expectation was that gene conversion would show a greater tendency to confine modification to the IgH coding sequence, leaving intron regions substantially unmodified. An analysis of rearranged IgH sequences from cattle of different ages revealed that the diversification of germline sequences could be observed in very young calves and that substitution frequency increased with age. The age-dependent accumulation of mutations was particularly apparent in the second IgH complementarity-determining region (CDR2). Single base substitutions were found to predominate, with purines targeted more frequently than pyrimidines and transitions favoured over transversions. In non-coding regions, mutations were detected at a normalised frequency that was indistinguishable from that observed in CDR2. These data are consistent with a process of IgH diversification driven predominantly by somatic hypermutation.     

荣昌猪白细胞介素-2受体γ链基因克隆、表达与鉴定

运用RT-PCR技术从由刀豆蛋白(ConA)诱导培养的荣昌猪外周血单核淋巴细胞(PBMC)扩增出猪白细胞介素-2受体γ链基因的完整开放阅读框(ORF),长约1 107 bp,编码由368个氨基酸残基组成的相对分子质量为41 780的蛋白多肽.荣昌猪IL-2Rγ与人、猕猴、牛、犬、鼠在氨基酸水平上的同源性分别为81.6%,80.8%,85.1%,83.2%和68.8%.运用PCR技术从含荣昌猪IL-2Rγ,开放阅读框序列质粒中扩增其成熟蛋白编码基因,共1 020 bp.将其定向克隆于原核表达载体pET-32a(+)后在E.coli BL21中诱导表达,SDS-PAGE结果显示表达的融合蛋白约为52 000,重组蛋白以包涵体的形式表达,表达产物约占茵体总蛋白的37.6%;Western-blotting分析表明,在相对分子质量52 000处有一奈特异性的带;对γ诱导重组茵进行转录检测得到约1 020 bp的特异性片段.     

Characterization of serum immunoglobulin M of grouper and cDNA cloning of its heavy chain

Immunoglobulin M (IgM) from the whole serum of grouper fish, Epinephelus coioides was purified by affinity chromatography using protein A-Sepharose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions revealed that the relative molecular masses (Mr) of the equimolar heavy and light chains of IgM were 78,000 and 27,000, respectively. The cDNAs encoding IgM heavy chain comprising its variable (VH) and constant (CH) regions have been cloned and sequenced from a grouper kidney cDNA library by antibody screening method. Five VH (130-142 amino acids) and four CH (450-454 amino acids) families were identified. The variable and constant regions were conserved with their putative domains. All the four constant region domains (CH1-CH2-CH3-CH4) contained each three conserved cysteine residues, which are considered to form the inter- and intra-chain disulfide bridges. There were three carbohydrate acceptor sites in the constant region. In general, the pattern of IgM gene organization seems to resemble that of other teleosts. Moreover, the CH genes in grouper IgM occur as multifamily as reported in Atlantic salmon and common carp.     

Partial sequence of Mongolian gerbil (Meriones unguiculatus) immunoglobulin gamma heavy chain constant region

We determined the sequence of the immunoglobulin gamma heavy‐chain constant (IGHC) region of the Mongolian gerbil (Meriones unguiculatus). To isolate a part of the IGHC complementary DNA, we designed primers on the basis of highly conserved sequences in mouse, rat and hamster. The deduced IGHC is structurally similar to counterparts in other mammalian species and shows 84.6% identity to the IGHC of hamster IgG, 76.6% to rat IgG1, 83.3% to rat IgG2a, 78.1% to mouse IgG1, 81.8% to mouse IgG2a, 79.1% to mouse IgG2b and 79.2% to mouse IgG3 at the nucleotide level. The results suggest that gerbil IgG is closely related to hamster IgG and rat IgG2a.     

Early, microbially driven follicular reactions in the neonatal piglet do not contribute to expansion of the immunoglobulin heavy chain V-D-J repertoire

Selective microbial colonisation of germ-free piglets is reported to result in expansion of immunoglobulin V(H)- and D(H)-segment usage from an initially limited repertoire. Here, the response of the palatine tonsil to microbial colonisation was compared in age-matched conventionally reared and germ-free piglets. At 3 and 5 days after birth an expansion in the B-cell follicle area was observed in the conventional, microbially colonised animals, which was not seen in the germ-free piglets. Consistent with this observation, sequencing of re-arranged heavy chain V-D-J units demonstrated accumulation of point mutations indicating somatic hypermutation in the conventional, microbially colonised piglets but not in the germ-free animals. However, V(H)- and D(H)-segment usage and CDR3 length did not differ between the groups. The results suggest that the follicle reaction observed occurs in response to microbial challenge, involves proliferation and somatic hypermutation of B-cells but does not expand repertoire or generate classical, isotype-switched memory B-cells. We suggest that microbial colonisation of neonatal piglets drives immunological competence in two stages: first, an antigen non-specific, follicular reaction which expands immunological compartments; and second, microbe driven changes in V-segment usage which expand immunological repertoire.     

Relationships among serum immunoglobulin concentration in foals, colostral specific gravity, and colostral immunoglobulin concentration

Postpartum, presuckle, colostrum samples were collected from 100 mares. Colostral specific gravities significantly correlated (r = 0.9) with colostral immunoglobulin (Ig)G concentrations. Foal serum IgG concentrations highly correlated (r = 0.82) with specific gravities of the colostrum each foal ingested. Eight of 48 foals (17%) had serum IgG concentrations less than 400 mg/dl. The dams of these 8 foals had colostral sp gr less than 1.06 and colostral IgG concentrations less than 3,000 mg/dl. Foals had serum IgG concentrations greater than 520 mg/dl 24 hours after parturition, when the colostral specific gravity of the dam was greater than or equal to 1.06. Effects of breed on colostral specific gravity, colostral IgG concentrations, foal serum IgG concentrations, and mare serum IgG concentrations were not significant.     

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus)

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.     

Monoclonal anti-equine IgE antibodies with specificity for different epitopes on the immunoglobulin heavy chain of native IgE

In this study we describe the generation of monoclonal antibodies (mAbs), which recognize different epitopes of the equine IgE constant heavy chain. Equi-murine recombinant IgE (rIgE), composed of the murine V(H)186.2 heavy chain variable region, linked to the equine IgE constant heavy chain and expressed together with the murine lambda(1) chain in J558L cells was used to immunize BALB/C mice. A total of 17 different mAbs were obtained, which recognized the rIgE heavy chain constant region. None of the mAbs reacted with monoclonal equine isotypes IgM, IgG1 (IgGa), IgG3 (IgG(T)), IgG4 (IgGb) or isolated equine light chains, IgGc and IgA from horse serum, or the native mAb B1-8delta, expressing the same heavy chain variable regions and light chains. One of the mAbs (alphaIgE-132) recognized the recombinant equine IgE, but did not recognize any protein in equine serum, i.e. native IgE. A total of 16 mAbs detected a serum protein of approximately 210,000Da on Western blots, corresponding to the expected MW of native IgE. In addition, one of the mAbs (alphaIgE-176) detected a protein of 76,000Da under reducing conditions, most likely the equine IgE heavy chain. According to binding inhibition studies, the equine IgE specific mAbs recognize at least two different epitopes of the equine IgE. In an ELISA using two anti-IgE mAbs which recognized different epitopes, no significant differences in the concentration of total serum IgE could be detected between adult Icelandic horses with IgE-mediated type I allergy (summer eczema) and healthy control animals. In Icelandic horse foals, no serum IgE could be measured 6 months post partum. All anti-IgE mAbs recognized a small population (1.3+/-0.5%) of leukocytes from adult Icelandic horses by surface immunofluorescence, but no cells could be detected in foal blood. The stained leukocytes from adult horses could be enriched by magnetic cell sorting and contained 32% basophils, 53% monocytes and/or large lymphocytes, 13% small lymphocytes and 2% eosinophils.     

Multiplex PCR and Genescan analysis to detect immunoglobulin heavy chain gene rearrangement in feline B-cell neoplasms

Lymphoid neoplasms are usually diagnosed on the basis of cytological and histopathological findings. However, in some cases, discrimination of lymphoid neoplasms from reactive lymphoid proliferation is difficult. PCR amplification of complementarity-determining region 3 (CDR3) of the immunoglobulin heavy-chain variable region (IGHV) gene can be used to assess clonality of B-cell populations as a supportive diagnostic tool for B-cell neoplasms. Because of the sequence variation and possible somatic hypermutation of the IGHV gene, sensitivity of the PCR-based assay to detect clonal IGHV gene rearrangement largely depends on the sequences and numbers of primer sets. Prior to the development of an efficient assay, we cloned and sequenced 97 IGHV complementary DNAs (48 IGHV-1 and 49 IGHV-3 clones) from normal cat spleens. On the basis of these sequences, we designed 6 forward primers at the variable region and 5 reverse primers at the joining region. Using each of 6 forward primers and a mixture of 5 reverse primers, we amplified CDR3 of IGHV genes and analyzed the PCR products by conventional PAGE and Genescan analyses using fluorescence-labeled primers. Twenty-six feline B-cell neoplasms diagnosed by histopathological and immunohistochemical examinations were subjected to the newly developed analysis of IGHV gene rearrangement. Clonal IGHV gene rearrangement was detected in 22 of 26 (84%) samples by both PAGE and Genescan analyses. To reduce the number of PCR reactions, we constructed a multiplex PCR analysis system using a mixture of IGHV-1- and IGHV-3-specific primers as forward primers and a mixture of 5 joining region reverse primers. Results of the multiplex PCR were 100% concordant with those obtained by each of the singleplex PCRs. The multiplex PCR-based assay and Genescan analysis developed in the present study would be useful and practical tools to detect clonal IGHV gene rearrangement in feline B-cell neoplasms.     

抗CDV、CAV卵黄抗体的制备

本研究用CDV、CAV分别免疫产蛋鸡，收集鸡蛋，用海藻酸钠—硫酸铵法提取卵黄抗体(IgY)，并对提取的IgY采用了SDS-PAGE和低压层析检测纯度，结果表明用该法提取的IgY纯度较高。用间接BUSA测定提取的IgY活性，结果证明提取的抗CAV IgY的活性较高，而抗CDV IgY的活性损失较大。     

Molecular cloning and characterization analysis of immunoglobulin M heavy chain gene in European eel (Anguilla anguilla)

In this study, the immunoglobulin M heavy chain gene of European eel (Anguilla anguilla) was cloned and analyzed. The full-length cDNA of the IgM heavy chain gene (GenBank accession no. EF062515) has 2089 nucleotides encoding a putative protein of 581 amino acids. The IgM heavy chain was composed of leader peptide (L), variable domain (VH), CH1, CH2, Hinge, CH3, CH4, and C-terminus and two novel continuous putative N-glycosylation sites were found close to the second cysteine of CH3 in A. anguilla-H1 and A. anguilla-H2. The deduced amino acid sequence of the European eel IgM heavy chain constant region shared similarities to that of the Ladyfish (Elops saurus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), Grass carp (Ctenopharingodon idella), Common carp (Cyprinus carpio), Channel catfish (Ictalurus punctatus), and the orange-spotted grouper (Epinephelus coioides) with the identity of 46.1%, 39.7%, 38.9%, 32.4%, 32.3%, 31.7%, and 30.7%, respectively. The highest level of IgM gene expression was observed in the kidney, followed by the spleen, gills, liver, muscle and heart in the apparently healthy European eels.     

鸡白细胞介素-2受体基因γ链的转录分析与表达

以脾单个核细胞总RNA为模板，对鸡白细胞介素-2受体基因γ链（chIL-2Rγ）进行了RT—PCR，获得了-1047bp的开放阅读框，编码由348个氨基酸残基组成的分子质量为37．8ku的蛋白多肽。预测的鸡IL-2Rγ多肽链中包含4个保守半胱氨酸残基、1个WSXWS基序和7个N连接的糖基化位点。鸡IL-2Rγ与其他动物IL-2Rγ在氨基酸水平上的同源性仅为21．49／6～38．2％。RT—PCR检测发现，鸡IL-2RγmRNA分布于大脑、小脑、脊髓、腔上囊、脾、胸腺、骨髓、盲肠扁桃体、腺胃、肌胃、空肠、卵黄囊憩室、回肠、盲肠、直肠、心脏、肾、肺、肝、骨骼肌和皮肤，而在十二指肠和睾丸中没有检测到其转录。构建了鸡IL-2Rγ胞外区的原核重组表达载体，进行了表达和鉴定。