人源性抗HIV—1 V3环噬菌体抗体的基因克隆与序列分析

以ＨＩＶ－１ ｇｐ１２０Ｖ３环的合成环肽为抗原,从人的噬菌体抗体组合文库中,筛选出与ＨＩＶ－１ Ｖ３肽具有结合活性的人源性噬菌体抗体,用ＥＬＩＳＡ测定其活性,竞争抑制实验证实了该噬菌体抗体的特异性。序列分析表明,重链基因的ＩｇＧ１亚类,可变区属ＶＨ Ｉ亚组,与胚系基因ＤＰ－８８的同源性最高。Ｄ区为Ｄ３－３,Ｊ区为ＪＨ５,轻链为ｋ亚型,可变区属ＶＬ ＩＶ亚群,Ｄ区为ＤＰＫ２２,Ｊ链为ＪＫ４胚系。     

从人源性噬菌体抗体库分离出1株含有异常序列的抗HBsAg Fab克隆

曾从人源性噬菌体抗体库中筛选出１株抗人乙肝表面抗原的Ｆａｂ我隆，为了筛选出新的抗ＨＢｓＡｇＦａｂ段，采用抗原屏蔽法，用已得到的Ｆａｂ段封闭相应的抗原决定基，对该抗体库进行了再次筛选，得到了１株新的人抗ＨＢｓＡｇＦａｂ段克隆经序列分析发现其轻链可变区基因来源于Ｖｋ１亚群和Ｊｋ４基因，重链可变区基因来源于ＶＨ１亚群和ＪＨ４基因，但在ＶＨ第７７位和第７８位氨基酸基之间出现了７个多余的氨基酸残基，经对基因     

AChR单抗(A7)诱导大鼠了实验性重症肌无力的分子基础

乙酰胆碱受体（ＡＣｈＲ）抗体在重症肌无力（ＭＧ）和实验性自身免疫性重症肌无力（ＥＡＭＧ）患者体内可诱导ＡＣｈＲ丢失和肌力减退。通过对小鼠抗电鳐电器官的ＡＣｈＲ单抗Ａ７的致病性测定和可变区基因克隆与序列分析，　探讨致病性抗体介导的ＭＧ和ＥＡＭＧ的发病机制。Ａ７被动注射大鼠后可诱导出严重的ＥＡＭＧ，ＡＣｈＲ损失串达３８．４％±７．２％。Ａ７的Ｈ链Ｖ区由小鼠ＰＣ７１８３胚系基因编码与Ｄ和ＪＨ４连接，与ＤＦＬ１６．２胚系Ｖ_Ｈ基因具有９３．７％的同源性。Ｌ链Ｖ区由小鼠Ｖ_Ｋ３组基因编码，与Ｊｋ２连接，与Ｖｋ２１Ｅ胚系基因具有９８．１％的同源性。     

小鼠BMP—McAb可变区基因的体外扩增,克隆与序列分析

从体外培养的ＢＭＰＭｃＡｂ杂交瘤中提取细胞总ＲＮＡ，反转录成ｃＤＮＡ。以ｃＤＮＡ为模板，利用两对根据免疫球蛋白重链与轻链可变区５′端序列和Ｊ区序列设计合成的引物，通过ＰＣＲ方法，扩增出抗体重链、轻链可变区基因片段（ＶＨ，ＶＬ）。将扩增产物分别克隆入ｐＵＣ１８，ｐＵＣ１９质粒，筛选出阳性克隆，利用双脱氧链终止法进行序列测定，经计算机分析，ＶＨ基因全长为３４５ｂｐ，编码１１５个氨基酸；ＶＬ基因全长为３１５ｂｐ，编码１０５个氨基酸。     

早产儿与足月儿免疫球蛋白重链可变区基因异型性

目的：分析和比较早产儿和足月儿免疫球蛋白重链可变区基因中的可变区基因片段、多样化基因片段（Ｄ）和连接区基因片段的使用,ＶＨ上的体突变,在ＶＨ－Ｄ和Ｄ－ＪＨ连接点插入的ＮＭＤＮ长度以及开放性阅读框（ＯＲＦ）等特征及其差异性。方法：７例胎龄为２５－３０周的足月儿脐带单核细胞ＤＮＡ被抽提,使用套式ＰＣＲ扩增ＩｇＨ基因,扩增产物被克隆筛选和测序。结果：在早产儿,共有２０个不同的ＶＨ被使用,其中ＤＰ７３（Ｖ     

抗HSV糖蛋白单克隆抗体重链可变区DNA的克隆及序列分析

作者从分泌具有高中和活性和高保护作用的抗ＨＳＶ型共同性糖蛋白Ｃ的鼠源性单抗的杂交瘤细胞１Ａ１２／４Ｄ５中提取ＲＮＡ，逆转录成ｃＤＮＡ。用合成的寡核苷酸引物从中扩增了抗体的重链可变区基因，并克隆入质粒，随机挑取两个阳性克隆进行核苷酸序列分析，见所克隆基因确可编码小鼠抗体的重链可变区。     

抗HSV糖蛋白单克隆抗体重链可变区DNA的克隆及序列…

作者从分泌具有高中和活性和高保护作用的抗ＨＳＶ型共同性糖蛋白Ｃ的鼠源性单抗的杂交瘤细胞１Ａ１２／４Ｄ５中提取ＲＮＡ、逆转录成ｃＤＮＡ。用合成的寡核苷酸引物从中扩增了抗体的重链可变区基因，并克隆入质粒，随机挑取两个阳性克隆进行核苷酸序列分析，见所克隆基因确可编码小鼠抗体的重链可变区。     

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF THE IMMUNOGLOBULIN LIGHT CHAIN VARIABLE REGION GENE OF A MURINE ANTI-HUMAN C1-INH IgG

获得抗体的可变区基因是由鼠源性ＭｃＡｂ制备重组抗体的前提，本实验合成与轻链可变区（ＶＬ）ＦＲ１和ＦＲ４互补的通用引物，由分泌抗人Ｃ１－ＩＮＨＭｃＡｂ的杂交瘤Ｆ７细胞株提取总ＲＮＡ，经ＲＴ－ＰＣＲ扩增出Ｆ７ＶＬ的ｃＤＮＡ片段，并将其克隆入ｐＵＣ１８／１９测序载体，从两端进行双脱氧核苷酸随机终止法的ＤＮＡ序列测定。结果显示：ＶＬｃＤＮＡ是由３０３个碱基组成，编码１０１个氨基酸残基。由国际联机检索进行ＥＭＢＬ和Ｋａｂａｔ基因库扫描发现：Ｆ７ＶＬ仅与Ｉｇ同源，符合小鼠Ｉｇ的ＶＬ基因特征，同源性为６０％～８０％。根据Ｋａｂａｔ分类方法，Ｆ７ＶＬ基因推导的氨基酸序列应归属于小鼠Ｉｇ的ＶＬ基因ＩＶ亚组，是由Ｖｋ－Ｊｋ１重排产生。ＣＤＲ３含有９个氨基酸残基，其中含３个不相同氨基酸残基，ＶＬ大多数的氨基酸变化集中在ＦＲ１和ＣＤＲ１区，Ｆ７ＶＬ符合ＩｇＶＬ氨基酸残基变化的基本特征。Ｃｙｓ２３和Ｃｙｓ８８残基位于Ｆ７ＶＬＣＤＲ１和ＣＤＲ３的起始点，与二硫键形成有关。成功获得Ｆ７ＶＬ基因为进一步构建和表达单链Ｆｖ（ｓｃＦｖ）抗体打下了良好的基础。     

随机化CDR3抗体库的构建及不经免疫制备抗体的初步探索

为探索利用噬菌体抗体库技术不经免疫制备抗体，在已有的抗乙肝病毒表面抗原（ＨＢｓ）人Ｆａｂ段基因的基础上，通过ＰＣＲ引入突变，对其ＶＨＣＤＲ３中１２个氨基酸残基（９５～１００Ｊ）进行了完全随机化，对９４和１００Ｋ位残基进行了有限随机化，构建了半合成噬菌体抗体库，库容为２×１０８，５８％含有随机化ＣＤＲ３序列，用无关抗原小鼠ＩｇＧ对该抗体库进行筛选，获得了三个可与小鼠ＩｇＧ结合而不与ＨＢｓ反应的克隆，经测定其ＣＤＲ３的ＤＮＡ序列，发现这三个克隆具有相同的ＣＤＲ３，证明单纯改变ＶＨＣＤＲ３可以得到新的特异性抗体，从而绕过免疫制备抗体。     

CLONING AND SEQUENCE CHARACTERIZATION OF THE RE-ARRANDED VH GENE FROM HYBRIDOMA CELLS SECRETING ANTI-HUMAN C1-INHIBITOR McAb

由分泌抗人Ｃ１－ＩＮＨＭｃＡｂ的杂交瘤Ｆ７细胞株提取总ＲＮＡ，合成与ＶＨ基因ＦＲ１和ＦＲ４互补的通用引物，以ＲＮＡ反转录合成的第一链ｃＤＮＡ为模板，ＰＣＲ法克隆出Ｆ７ＶＨ基因的ＤＮＡ片段。将分离获得的目的ＤＮＡ片段亚克隆入ｐＵＣ１８／１９测序载体，从两头进行双脱氧核苷酸随机终止法的ＤＮＡ序列测定。结果显示：ＶＨ基因是由３３３个碱基组成，编码１１１个氨基酸残基。通过国际联机检索进行ＥＭＢＬ和Ｋａｂａｔ基因库扫描发现，Ｆ７ＶＨＤＮＡ仅与Ｉｇ同源，符合小鼠Ｉｇ的ＶＨ基因特征；同源性为６０％～８５％，应归属于Ｉｇ的ＶＨ基因。根据Ｋａｂａｔ分类方法，Ｆ７ＶＨ基因推导的氨基酸顺序属于小鼠Ｉｇ的ＶＨ基因的Ⅱ（Ａ）亚组，是由ＶＨ－Ｄ－ＪＨ３重排产生；其框架区的９和６７位点为脯氨酸（Ｐｒｏ）和赖氨酸（Ｌｙｓ）（非芳香族氨氨酸），符合Ⅱ（Ａ）亚组框架残基结构模式；其ＣＤＲ３区含有７个氨氨酸残基，表明Ｃ１－ＩＮＨ抗原表面结构并不复杂；ＦＲ２和ＦＲ３的２２和８９位点为半胱氨酸残基（Ｃｙｓ），与ＶＨ片段二硫键形成有关。成功获得Ｆ７ＶＨ基因为进一步构建和表达单链Ｆｖ（ｓｃＦｖ）抗体打下良好的基础。     

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.     

从半合成噬菌体抗体库中筛选抗角蛋白人抗体

目的：从半合成噬菌体抗体库中筛选人源性抗角蛋白抗体并进行鉴定。方法：以表皮角蛋白为抗原,通过吸附－洗脱－扩增过程从半合成噬菌体抗体库中筛选特异性抗角蛋白抗体,对其抗原结合活性的序列进行分析鉴定。结果;经过４轮筛选,获得２０个能与角蛋白结合的阳性克隆,其中可产生特异性抗角蛋白抗体的克隆１８个。经ＤＮＡ指纹分析,判断所获克隆分别包含４个不同的Ｆａｂ段基因。     

Molecular analysis of HIV-1 gp120 antibody response using isotype IgM and IgG phage display libraries from a long-term non-progressor HIV-1-infected individual.

To characterize the variable heavy chain (VH)3 antibody response to HIV-1 gp120, we analyzed a panel of IgM and IgG1 Fab fragments from phage display isotype libraries from a long-term, non-progressor HIV-1-infected individual. The IgM Fab antibodies isolated had low affinity for gp120, were not restricted to a particular VH3 germ-line gene, and consisted mainly of unmutated VH genes. In contrast, IgG Fab fragments were gp120 specific, with high affinity and extensive somatic mutation; all were clonally related and were derived from a single VH3 germ-line gene (DP50). One IgG Fab (S8) has DP50 VH region nucleotide substitutions identical to those of IgM Fab M025 and uses similar DH and JH segments, suggesting that S8 arose from M025 by isotype switching. In addition, somatic mutation in the IgG heavy chain third complementarity-determining region results in a 100-fold affinity increase for gp120, which correlates with a similar increase in neutralization capacity. These results imply that in vivo IgM to IgG isotype switch and affinity maturation may be important for protection and long-term survival in certain HIV-1-infected individuals.     

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.     

Production of high-affinity human monoclonal antibody fab fragments to the 19-kilodalton C-terminal merozoite surface protein 1 of Plasmodium falciparum

A combinatorial immunoglobulin gene library was constructed from peripheral blood lymphocytes of eight patients infected with Plasmodium falciparum and was screened for the production of human monoclonal antibody Fab fragments to the C-terminal 19-kDa fragment of P. falciparum merozoite surface protein 1 (MSP-1(19)). Three Fab clones recognized recombinant MSP-1(19) under nonreducing conditions. Indirect immunofluorescence microscopy demonstrated that three Fab clones stained the surfaces of late trophozoites/schizonts and merozoites of the FCR3 and 3D7 strains, suggesting the Fabs' reactivities to a conserved epitope. Sequence analysis of the heavy-chain genes revealed that the closest germ line V segments were VH1-8 and VH7-81, with 91% to 98% homology. The closest germ line D segment was D3-10, and the closest germ line J segment was JH4 or JH5, with 90% to 97% homology. In the light-chain genes, the closest germ line V segment was A27 for the Jkappa2, Jkappa4, and Jkappa5 segments. The dissociation constants of these Fab fragments for recombinant MSP-1(19) ranged from 1.09 x 10(-9) to 2.66 x 10(-9) M. The binding of the three Fab fragments to MSP-1(19) was competitively inhibited by the anti-MSP-1(19) mouse monoclonal antibody 12.8, which inhibits erythrocyte invasion by merozoites. However, the human Fab fragment with the highest affinity did not inhibit in vitro growth of P. falciparum. This is the first report of gene analysis and bacterial expression of human monoclonal antibodies to P. falciparum MSP-1(19). The combinatorial immunoglobulin gene library derived from malaria patients provides a potential tool for producing high-affinity human antibodies specific for P. falciparum.     

VH3 gene usage in neutralizing human antibodies specific for the Entamoeba histolytica Gal/GalNAc lectin heavy subunit

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomatic Entamoeba histolytica cyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-specific lectin of E. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the V kappa 1 family, in which the closest V kappa germ line gene was 02/012 or L5, with the J kappa 1, J kappa 2, J kappa 4, or J kappa 5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation of E. histolytica trophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.     

Characterization of a human monoclonal autoantibody directed to cardiolipin/beta 2 glycoprotein I produced by chronic lymphocytic leukaemia B cells.

We determined the specificity and sequence of immunoglobulin molecules synthesized by monoclonal B cells from a patient with chronic lymphocytic leukaemia (CLL) who presented with a number of clinical and biological autoimmune symptoms. Heterohybrids obtained by fusion of CLL cells with the mouse X63-Ag 8.653 myeloma produced IgM lambda MoAbs directed to the cardiolipin/beta 2 glycoprotein I (beta 2GPI) complex and ssDNA. They were devoid of polyreactivity. Nucleotide sequence analysis of the variable domain of the mu chain indicated the utilization of the VH4 71.2 gene or one allotypic variant, DXP4 and JH3 segments. The lambda light chain used the single gene from the V lambda 8 subfamily, J lambda 3 and C lambda 3 genes. The VH gene displayed 11 nucleotide changes in comparison with its putative germline counterpart. However, these nucleotide changes correspond to variations observed in other published VH4 sequences, suggesting gene polymorphism rather than somatic mutation. DXP4 and JH3 were also in germline configuration. The VL gene exhibited a single replacement mutation in CDR1. These data suggest that the monoclonal CLL B cells in this patient retained VH and VL genes in germline configuration although they secreted a pathogenic anti-cardiolipin antibody associated with clinical symptoms, vasculitis and thrombosis, which may be provoked by antibodies to the phospholipid/beta 2GPI complex.     

人源性抗HBsAg单链Fab基因在Pichia pastoris中的表达

目的：研究重组Fab的结构与亲和活性的关系：方法：通过重叠PCR，将人源性抗HBsAg Fab的H和L链基因融合构建单链Fab基因，并将其转入毕赤酵母表达载体pPICZαA中。以单链Fab基因表达载体通过氯化锂转化法转化毕赤酵母GS115。将获得的重组酵母在摇瓶中培养进行重组单链Fab的可溶性表达。表达上清经硫酸铵沉淀及亲和层析纯化后，用直接ELISA检测表达产物和纯化Fab的活性：结果：SDS—PAGE和Western blot分析显示，单链Fab在毕赤酵母中获得分泌型表达。薄层扫描显示，在摇瓶中培养毕赤酵母表达的单链Fab约为5～10mg／L。经亲和层析纯化获得纯度达97．8％的重组单链Fab。经直接ELISA测定的结果显示，重组单链Fab具有较好的结合HBsAg的活性。结论：通过重叠PCR构建的融合单链Fab基因，可成功地在毕赤酵母中获得分泌型表达，表达产物具有较好的结合HBsAg的活性。     

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.