新生儿免疫球蛋白重链可变区基因多样性分析

目的构建不同胎龄新生儿IgH基因指纹图谱,探讨新生儿成熟度对IgH基因谱型多样性的影响.方法提取27～42周新生儿RNA、进行RT-PCR反应、6%变性聚丙烯酰胺凝胶电泳和银染显色,获得不同胎龄新生儿IgH基因指纹图谱,用Tiger凝胶图像分析仪进行曲线转化和分析.结果各家族指纹图谱转化后的曲线下面积积分值,在28～32周、33～36周和37～42周新生儿之间无差异;但胎龄27周新生儿曲线下面积小于胎龄28～42周新生儿.结论 28周可能是人类IgH基因谱型发育由不完善到趋于完善的一个转折点,28周到足月的新生儿IgH基因谱型发育趋于稳定.     

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     

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。     

用基因定点突变技术改建—免疫球蛋白重链可变区基因的通用表达载体

本文介绍了一种简便的、具有高突变率的基因定点突变方法。应用Bio-Rad Muta-Gene Phagemid Kit,在一装有表达载体上的抗NP(4-hydroxyl-3-nitrophenacetyl)免疫球蛋白重链可变区基因的J_a区引进BstE Ⅱ酶切位点,从而改建成一种能用于表达免疫球蛋白重链可变区基因的通用表达载体。应用该Kit,基因定点突变率达43％。     

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.     

两株人单抗重链可变区基因特征分析

对采用PCR方法克隆获得的两株抗出血热病毒人单抗重链可变区基因(V_H87—2,V_HC8)进行了基因特征分析。同源性比较显示:两基因来源于不同的人抗体重链可变区家族。V_H87-2与人免疫球蛋白重链可变区第三亚组同源性最高,达87％,而V_HC8与人免疫球蛋白重链可变区第一亚组同源性最高,达71％。空间结构分析表明两个可变区具有不同空间构象:V_H87—2属人抗体重链可变区1—1类空间结构,V_HC8属人抗体重链可变区1—2类空间结构。上述分析证明,此两株人单抗重链可变区存在着基因特征及蛋白空间构象的差异。通过基因分析掌握人单抗分子特征,对指导人源性基因工程抗体的研究具有重要意义。     

单克隆抗体3D3重链可变区基因结构分析

单克隆抗体（ＭｃＡｂ）在科研及影像诊断等领域应用十分广泛，鼠源性单克隆抗体，由于其抗原性，在人体疾病治疗应用受到限制，利用ＤＮＡ重组技术，构建各种重组抗体基因，表达制备重组抗体，可降低其抗原性，前提是必须获得其可变区基因，我们利用设计合成的一对鼠抗体...     

抗人ClqMcAb轻、重链可变区基因的克隆、序列分析及轻链可变区基因的表达（摘要）

抗人ＣｌｑＭｃＡｂ轻、重链可变区基因的克隆、序列分析及轻链可变区基因的表达（摘要）陈克敏，朱锡华应用ＰＣＲ技术，从一株抗人ＣｌｑＭｃＡｂ杂交瘤细胞基因组ＤＮＡ及反转录合成ｃＤＮＡ中，扩增、克隆分别获得抗人Ｃｌｑ轻、重链可变区基因，序列分析表明：抗人Ｃ...     

抗HLA－A2单抗重链可变区基因的克隆及其序列分析

从５株抗ＨＬＡ－Ａ２单抗杂交瘤细胞株中提取ｍＲＮＡ，以特异性引物采用ＰＣＲ技术，扩增和克隆出单抗可变区重链基因，并采用双脱氧链式终止法及计算机基因文库测定和分析了其全部的氨基酸序列，对抗体的生物学特性的研究具有重要意义，为构建基因工程抗体奠定了基础。     

抗BAFF单克隆抗体轻链和重链可变区基因的克隆及鉴定

目的:在本实验室成功制备小鼠抗人BAFF单克隆抗体的基础上,通过分子克隆方法获得该抗体可变区基因序列。方法:从1株小鼠抗人BAFF单克隆抗体杂交瘤细胞FMMUB4中提取总RNA,以此为模版反转录成cDNA,用针对小鼠单克隆抗体重链和轻链可变区基因序列的特异性引物分别进行PCR反应,PCR产物连接入载体,经筛选阳性克隆、酶切鉴定后送测序,测序结果进行生物信息学分析。结果:成功克隆了抗BAFF单克隆抗体FMMUB4重链和轻链可变区基因,测序结果证实序列正确。结论:获得了抗BAFF单克隆抗体FMMUB4重链和轻链可变区基因序列,为下一步构建相关基因工程抗体打下良好基础。     

抗端粒酶蛋白hTERT重链可变区基因的克隆和表达

利用噬菌体表面展示技术制备端粒酶蛋白hTERT抗体，从重组的hTERT免疫的小鼠脾淋巴细胞中提取总RNA，逆转录成cDNA，以逆转录合成的cDNA第一条链为模板，用VHback和VHfor引物，通过PCR扩增出约350bp大小的抗hTERT重链可变区基因，将其克隆到噬菌粒载体PCANTAB 5E中，转化入E.coli TG1宿主菌后，在M13K07辅助噬菌体帮助下，将VH与g3蛋白形成的复合物展示于噬菌体表面，我们利用Dot Blot检测方法筛选出hTERT具有亲和性的VH单功能区抗体，结果提示噬菌体展示技术是制备抗体的一种有效的新途径。抗hTERT VH基因克隆成功，为进一步构建单链抗体SvFv奠定了基础。     

Relation Between the Heavy Chain Complementarity Region 3 Characteristics and Rheumatoid Factor Binding Properties

Among the rheumatoid factors (RFs), monospecific and polyspecific types can be distinguished. However the molecular basis responsible for their different specificity is not well understood. In a previous report, we have shown that the binding of the majority of the polyspecific antibodies is salt-sensitive. No binding to IgG was observed under high ionic strength (0.3-0.5 M NaCl). This salt-sensitivity was only observed for 18% of the monospecific RFs. Here, we have analyzed 14 RFs representing the 3 different groups (6 salt-insensitive monospecific, 4 salt-sensitive monospecific and 4 salt-sensitive polyspecific RFs). By analysis of the amino acid composition and the distribution of polar and non-polar residues of their heavy chain complementarity-determining region 3 (H-CDR3) in relation to mono/polyspecifieity, salt-sensitivity and reactivity against human IgG subclasses, we have identified common structural features responsible for their different binding properties. Salt-sensitive RFs (mono as well as polyspecific antibodies) were characterized by long H-CDR3′s (15.3 ± 2.7) that contained large numbers of hydrophilic residues such as arginine and serine, while salt-insensitive RFs had more hydrophobic H-CDR3′s of smaller length (11.3 ± 2.4). In addition, for the monospecific RFs, remarkably similar hydrophilicity H-CDR3 profiles were found that were correlated with their specificity for IgG subclasses. These observations confirm the importance of the H-CDR3 for the binding of RFs to IgG. Furthermore, on the basis of their shorter H-CDR3′s and their rather unique H-CDR3 hydrophilicity profiles, it is likely that the majority of the monospecific RFs should be considered as a group of RFs that is independent of the polyspecific RF repertoire.     

Human Heavy Chain Variable Region Gene Diversity,Organization, and Expression

Elucidation of the cellular and molecular mechanisms which determine the expressed antibody repertoire remains a major challenge in immunology. Knowledge of V gene diversity, organization, and expression is important to an understanding of the formation of the antibody repertoire in normal as well as diseased states. In the last few years, great advances have been made in our understanding of the human heavy chain variable region (V_H) gene locus. In this review we present the current knowledge of V_H gene diversity, organization, and utilization in normal individuals followed by a discussion of the possible relevance of these findings to autoimmunity.     

IgTree: creating Immunoglobulin variable region gene lineage trees

Lineage trees describe the microevolution of cells within an organism. They have been useful in the study of B cell affinity maturation, which is based on somatic hypermutation of immunoglobulin genes in germinal centers and selection of the resulting mutants. Our aim was to create and implement an algorithm that can generate lineage trees from immunoglobulin variable region gene sequences. The IgTree((c)) program implements the algorithm we developed, and generates lineage trees. Original sequences found in experiments are assigned to either leaves or internal nodes of the tree. Each tree node represents a single mutation separating the sequences. The mutations that separate the sequences from each other can be point mutations, deletions or insertions. The program can deal with gaps and find potential reversion mutations. The program also enumerates mutation frequencies and sequence motifs around each mutation, on a per-tree basis. The algorithm has proven useful in several studies of immunoglobulin variable region gene mutations.     

B淋巴细胞增生性疑难病例中IgH基因克隆性重排的分析

目的 探讨IgH基因克隆性重排对B淋巴细胞增生性疑难病变的辅助诊断价值.方法 检测77例B淋巴细胞增生性疑难病例中IgH基因的克隆性重排情况,均采用BIOMED-2系统IgH克隆性试剂盒中FR1、FR2、FR3三组家族引物进行PCR及聚丙烯酰胺凝胶电泳,硝酸银染色后观察,并对照最终病理诊断进行分析.结果 77例病变的最终病理诊断:B淋巴细胞反应性增生12例,不能排除B淋巴细胞不典型增生或淋巴瘤20例,B细胞性淋巴瘤45例.三组中FR1、FR2和FR3至少有一个为阳性的比值分别为2/12、11/20(55%)和36/45(80%).B细胞性淋巴瘤中,FR1、FR2和FR3的阳性率分别为60%(27/45)、60%(27/45)、56%(25/45),其类型有边缘区B细胞性淋巴瘤20例(其中黏膜相关淋巴组织型结外边缘区淋巴瘤18例,结内边缘区淋巴瘤2例),弥漫性大B细胞淋巴瘤7例,滤泡性淋巴瘤7例,套细胞性淋巴瘤1例,Burkitt淋巴瘤1例,浆细胞瘤4例,不能分型5例.FR1、FR2和FR3三者检测均为阴性但仍诊断为淋巴瘤9例(20%),其中1例后来出现肝脏B细胞淋巴瘤.对IgH基因重排阳性的B淋巴细胞反应性增生和不典型增生14例的随访结果,4例重新取活检后诊断为B细胞性淋巴瘤,其中3例IgH基因重排检测为阳性.结论 联合检测IgH基因FR1、FR2和FR3克隆性重排对B淋巴细胞增生性疑难病变诊断有重要的辅助价值;对形态改变和免疫表型诊断淋巴瘤依据不足而基因重排阳性者,重取活检或随访有一定价值;对阴性病例有必要补充IgH基因重排及IgK和IgL基因重排的检测以提高检测敏感性.