健康人Ig/C3双特异性免疫复合物与体液免疫的相关性

采用直线相关分析法,较系统地研究了健康人Ｉｇ／Ｃ３双特异性循环免疫复合物（ＩｇＭ／Ｃ３－ＴＣＩＣ、ＩｇＧ／Ｃ３－ＴＣＩＣ和ＩｇＡ／Ｃ３－ＴＣＩＣ）与ＩｇＭ、ＩｇＧ、ＩｇＡ和Ｃ３的相关性,比较了Ｉｇ／Ｃ３－ＴＣＩＣ和Ｃ３／Ｉｇ－ＴＣＩＣ分别与有关体液免疫指标关系的异同。结果发现,ＩｇＭ／Ｃ３－ＴＣＩＣ与总ＩｇＭ和总ＩｇＡ／Ｃ３－ＴＣＩＣ与总ＩｇＭ、总ＩｇＭ、总ＩｇＧ、总ＩｇＡ、和复合ＩｇＧ以及总Ｃ３     

健康人C3/Ig双特异性免疫复合物与体液免疫的相关性

采用直线相关分析法,较系统地研究了健康人 Ｃ３／ Ｉｇ 双特异性循环免疫复合物（ Ｃ３／ Ｉｇ Ｍ  Ｔ Ｃ Ｉ Ｃ、 Ｃ３／ Ｉｇ Ｇ Ｔ Ｃ Ｉ Ｃ 和 Ｃ３／ Ｉｇ Ａ Ｔ Ｃ Ｉ Ｃ）与 Ｉｇ Ｍ 、 Ｉｇ Ｇ、 Ｉｇ Ａ 和 Ｃ３ 的相关性．结果发现, Ｃ３／ Ｉｇ Ｍ  Ｔ Ｃ Ｉ Ｃ 和 Ｃ３／ Ｉｇ Ｇ Ｔ Ｃ Ｉ Ｃ 含量主要取决于血清总 Ｉｇ Ｍ 和 Ｉｇ Ｇ 量; Ｃ３／ Ｉｇ Ｇ Ｔ Ｃ Ｉ Ｃ 与复合的 Ｉｇ Ｍ 、复合的 Ｉｇ Ｇ、总的 Ｃ３ 呈正相关,而与复合的 Ｃ３ 呈负相关．这些结果提示, Ｉｇ Ｇ 在健康人体液免疫中的重要作用．同时说明,健康人机体免疫反应是复杂多样的,深入研究 Ｃ３／ Ｉｇ Ｔ Ｃ Ｉ Ｃ 与体液免疫的相关性及其形成机理,将为揭示健康人的体液免疫机制提供重要参考．     

抗人红细胞单链抗体基因的构建及表达

从分泌抗人红细胞单克隆抗体的杂交瘤细胞３Ｆ５中提取总ＲＮＡ,逆转录成ｃＤＮＡ,用合成的寡核苷酸引物通过ＰＣＲ扩增和克隆单抗的轻、重链可变区基因,用双脱氧核苷酸链终止法分析核苷酸序列,采用ＰＣＲ拼接法构建单链抗体基因,并插入原核表达载体ＰＥｔ－２８ａ,转化大砀杆菌ＢＬ２１（ＤＥ３）,经ＩＰＴＧ诱导后表达,序 分析表明所克隆的ＶＬ、ＶＨ分别属于鼠ＩｇＫａｐｐａ轻链ＩＶ亚组和Ｉｇ重链Ⅱ亚组,ＶＨ、ＶＬ基     

抗人前列腺素D合成酶嵌合抗体基因的构建

采用RT-PCR技术,从1株稳定分泌抗人L-PGDS单克隆抗体的鼠杂交瘤细胞中扩增抗体的可变区基因,得2个Vk和1个VH．序列经Igblast比对分析,其中1个VK为鼠骨髓瘤细胞系内固有的无功能基因;另-Vk和VH具备鼠抗体可变区特征,为抗体功能基因．经双酶切,将抗体可变区功能基因分别与表达载体pAG4622、pAH4604中的人IgG相应恒定区相连,构建成抗人L-PGDS的人-鼠嵌合抗体基因。     

E.coli和Yeast基因起始与终止密码子邻近序列碱基保 …

计算Ｅ．ｃｏｌｉ和Ｙｅａｓｔ基因起始与终止密码子邻近序列单碱基、相邻双碱基、相邻三碱基的碱基出现概率得出的Ｍ１（ｌ）、Ｍ２（ｌ）、Ｍ２（ｌ）值,很好地体现了原核生物Ｅ．ｃｏｌｉ和真核生物Ｙｅａｓｔ翻译起始区域的显著差异;矩阵Ｐ的本征值之和,可作为衡量不同生物基因碱基保守性,关联性强弱强度的一个指标。     

人源性抗HBc单链噬菌体抗体库的构建

利用RT－PCR和噬菌体表面展示技术，直接从乙肝病毒核心抗体（抗HBc）阳性患淋巴细胞中提取总RNA，反转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体（ScFv）基因，重组于噬菌粒载体pHEN1，转化抑制型大肠杆菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。库容量达106。     

抗转铁蛋白受体单抗可变区基因克隆和序列分析

目的:制备基因工程抗体,减少或消除鼠源性单抗在人体内的免疫原性,保留其对人体抗原配体的高度特异性,发展临床导向诊断和治疗。方法:从体外分泌抗转铁蛋白受体单抗杂交瘤细胞系7579中,对其单抗可变区基因进行克隆和序列分析。利用逆转录PRG技术扩增轻、重链可变区基因,再分别与pGEM-T载体连接、并克隆于JM109受体菌之中。利用荧光染色链终止法测定其序列,采用DNASIS7分析软件和与NIH基因库比较分析。结果:轻、重链可变区分别由139和128氨基酸残基组成,包括信号作、互补决定区和骨架氨基酸残基等功能序列。分别属于鼠免疫球蛋白重连Ⅱc和k链Ⅵ家族。结论:来自单抗的可变区的基因是完整的和具有潜在功能性的,为体外获得抗转铁蛋白受体基因工程抗体奠定了基础。     

乌鳢（Channa argus）干扰素刺激基因Viperin和ISG15及其启动子的克隆与特征分析

应用抑制差减杂交、末端快速扩增和基因步移技术,克隆并鉴定了乌鳢干扰素刺激基因Viperin和ISG15及其启动子序列.Viperin cDNA全长1474nt,包含1059nt的开放阅读框,编码352个氨基酸.除N末端70个氨基酸外,Viperin蛋白氨基酸序列在鱼类和哺乳类具有高度的保守性.ISG15 cDNA全长758nt,包含468nt的开放阅读框,编码155个氨基酸,含有两个泛素样(UBL)结构域,C末端具有保守的UB偶联结构(LRGG).Viperin和ISG15启动子区存在保守的干扰素刺激反应元件(ISRE)和γ-干扰素激活序列(GAS).ISRE是干扰素刺激基因启动子区的重要特征,并与干扰素调控因子(IRF)1和IRF2的识别序列(IRF-E)部分重复;GAS参与介导II型干扰素激活基因的诱导转录.Viperin启动子区还包含保守的NF-κB结合位点,这是保守的NF-κB结合位点以一致的序列模式(GGGRNNYYCC)出现在鱼类干扰素刺激基因启动子区的首次报道.此外,Viperin和ISG15启动子尚存在TATA,CAAT,Sp1等转录因子结合位点.乌鳢ISG15基因5′非编码区含有单一的内含子,而Viperin基因5′非编码区未发现内含子.Viperin和ISG15mRNA主要表达在头肾、后肾、脾脏和鳃,肝脏中也有少量表达.体内干扰素诱导剂polyI:C刺激后,Viperin和ISG15基因在肝脏的表达水平明显增加.     

鱼类免疫球蛋白分子生物学研究进展

综述了近年来鱼类免疫球蛋白分子生物学的最新研究进展,主要涉及鱼类Ig基因序列分析、组成形式、重排机制以及转录调控4个方面.鱼类Ig重链和轻链基因克隆分析证实有多种不同的重链、轻链类型存在;重链和轻链基因由不同染色体上的多基因座编码,在不同的鱼类中具有不同的基因组织形式;重组信号序列在重链、轻链基因组中均有发现,其重排过程主要由重组活化酶进行调控;增强子和启动子在鱼类Ig转录调控中发挥重要作用,其中增强子序列在系统发育中具有保守性.Ig作为鱼体特异性体液免疫应答的主要因子,其分子生物学的深入研究对于阐明脊椎动物免疫系统进化具有重要意义.     

鳜免疫球蛋白轻链基因在大肠杆菌中的表达

通过RT-PCR技术从鳜(Siniperca chuatsi)头肾总RNA中获得了免疫球蛋白轻链cDNA,克隆到pGEM-T载体上并测序.测序的2个克隆其可变区cDNA序列相同率为97.3%,属于鳜的同一个轻链可变区基因家族,其变异主要存在于互补性决定区.根据鳜与其他辐鳍鱼类轻链氨基酸序列的多重对准,鳜同鲑(Salmo salar)的可变区的相似性最高(65.7%).而在恒定区,鳜同花狼(Anarhichas minor)的相似性最高(96.3%),都存在特有的连续的丝氨酸残基;鳜同虹鳟(On-corhynchus mykiss)L1型(62.1%)和斑点叉尾(Ictalurus punctatus)G型(57.2%)轻链也有较高的相似性.将鳜轻链基因分别克隆到原核表达载体pET-28a(+)和pExSec1上,在大肠杆菌BL21(DE3)中以包涵体形式得到高效表达.另外,结合鳜轻链基因序列及其表达产物的分析结果,推断鳜也有两类轻链.     

Unusual sequences in the murine immunoglobulin mu-delta heavy-chain region

The delta heavy (H) chain of mouse immunoglobulin D (IgD) is unusual both in its structure and in its differential expression relative to immunoglobulin M (IgM; reviewed in ref. 1). The region of DNA between IgM and IgD H-chain constant-region genes is probably implicated in this control. So far only fragments of the area have been sequenced. Now, however, we present the complete sequence as well as the sequence of the introns of the C delta gene. We have found several interesting features (Fig. 1), including an open reading frame (ORF) between Cmu and C delta which encodes 146 amino acids that might represent a previously unsuspected domain-like protein; three blocks of simple repetitive sequences; a 162-base pair (bp) unique-sequence inverted repeat; and a domain-like pseudogene in the large intron of C delta. We have not found, however, any sequence 5' of C delta resembling the switch (S) recombination sequences associated with class switching in other heavy chains. Moreover, we have determined the 3' deletion end point of an IgD-producing myeloma and find no sequences reminiscent of switch sites nearby.     

Circular DNA is a product of the immunoglobulin class switch rearrangement

The class of immunoglobulin is defined by the constant region of its heavy chain. When a B lymphocyte switches the class of heavy chain it produces, the constant region of mu-type heavy chain is replaced; this occurs through a DNA rearrangement that brings the gene segment encoding the new constant region close to the VDJ segment encoding the variable region. The pre-B-cell line 18-81, which switches from heavy chain mu to gamma 2b production in culture, occasionally abnormally rearranges the heavy chain locus so that DNA sequences between the switch regions of mu and gamma 2b are inverted. Because looping-out is an intermediate step in generating an inversion, the switch rearrangement could occur by looping-out and deletion. Provided that recombination is reciprocal, this would produce a circle of DNA. Indeed, circular DNA molecules have been isolated as products of rearrangement among gene segments encoding the variable regions of the T-cell receptor and of the immunoglobulin heavy chain and light chain. But whereas the breakpoints for the variable region rearrangement are precisely defined, the breakpoints for any given heavy chain class switch are scattered over a length of greater than 6 kilobases, including both switch regions. We have now isolated circular DNA containing the sequences deleted by class-switching, thereby showing that the immunoglobulin heavy chain class switch occurs through looping-out and deletion.     

Mouse Cmu heavy chain immunoglobulin gene segment contains three intervening sequences separating domains

The IgM molecule is composed of subunits made up of two light chain and two heavy chain (mu) polypeptides. The mu chain is encoded by several gene segments--variable (V), joining (J) and constant (Cmu). The Cmu gene segment is of particular interest for several reasons. First, the mu chain must exist in two very different environments--as an integral membrane protein in receptor IgM molecules (micrometer) and as soluble serum protein in IgM molecules into the blood (mus). Second, the Cmu region in mus is composed of four homology units or domains (Cmu1, Cmu2, Cmu3 and Cmu4) of approximately 110 amino acid residues plus a C-terminal tail of 19 residues. We asked two questions concerning the organisation of the Cmu gene segment. (1) Are the homology units separated by intervening DNA sequences as has been reported for alpha (ref. 5), gamma 1 (ref. 6) and gamma 2b (ref. 7) heavy chain genes? (2) Is the C-terminal tail separated from the Cmu4 domain by an intervening DNA sequence? If so, DNA rearrangements or RNA splicing could generate hydrophilic and hydrophobic C-terminal tails for the mus and micrometer polypeptides, respectively. We demonstrate here that intervening DNA sequences separate each of the four coding regions for Cmu domains, and that the coding regions for the Cmu4 domains and the C-terminal tail are directly contiguous.     

The immunoglobulin mu constant region gene is expressed in mouse thymocytes

It has been a matter of controversy whether the functional capacity of T cells to discriminate between antigens is mediated via immunoglobulin, an immunoglobulin-like molecule, or by the product(s) of unrelated genes. The progenitors of immunoglobulin-secreting cells, B cells, express membrane-bound immunoglobulin as the antigen-specific receptor on their surface. For T cells, although products of immunoglobulin heavy chain variable region genes are implicated as receptor components, there has been no compelling immunochemical evidence for participation of either immunoglobulin light chains or heavy chain constant regions (see refs 2-6 for the disparate views). Recently, using cloned immunoglobulin DNA sequences as hybridization probes, we have demonstrated that the immunoglobulin Cmu gene, but not the Cmu gene, is expressed as polyadenylated RNA in some T cell tumour (T lymphoma) cell lines. Individual T lymphoma lines yielded up to three discrete mu RNA species of different sizes (1.9, 2.2 and 3.0 kilobases), each species being different in size from the major mu RNA species present in B lymphoma cells (2.4 and 2.7 kilobases). We show here that cells from the normal mouse thymus contain mu RNA species, indistinguishable in size from those in T lymphoma cells, but contain little if any kappa RNA.     

Critical test of a sister chromatid exchange model for the immunoglobulin heavy-chain class switch

B lymphocytes may switch from producing an immunoglobulin heavy chain of the mu class to that of the gamma, epsilon or alpha class. To maintain the specificity, the new heavy chain must keep the original variable (V) region; this is achieved by deleting DNA sequences so that the V (consisting of joined VH, diversity (DH) and joining (JH) gene segments) and C (constant) gene segments coding for the new heavy chain are brought into close proximity (reviewed in ref. 5; we do not consider here the mu-delta situation). There are, in principle, three types of chromosomal rearrangements that yield a deletion: rearrangement within a chromatid; unequal sister chromatid exchange (as suggested by Obata et al.); and unequal recombination between chromosomal homologues. We have analysed the arrangement of C mu DNA in clones of the pre-B-cell line 18-81 that switches in vitro from mu to gamma 2b. The clones examined produce either mu, gamma 2b or no immunoglobulin chain. We report here that all the gamma 2b clones had lost at least one copy of C mu and no clones contained three copies of C mu. These findings formally exclude both unequal sister chromatid exchange and recombination between homologues as mechanisms for creating a gene encoding the gamma 2b chain.     

两种抗α毒素单链抗体基因的序列分析

应用RT－PCR技术,从两株分泌具有中和活性的抗A型产气荚膜酸菌α毒素单克隆抗体（McAb)的杂交瘤细胞株2E3和1A8中,分别扩增出抗体VH和VL基因,用Linker(Gly4Ser）3基因,将VH和VL基因连接成ScFv基因2E3－ScFv和1A8－ScFv,并将其克隆至pGEM-T载体中,经核苷酸序列分析证实,VH和VL基因以及Linker基因拼接正确,2E3－ScFv基因全长为729bp,经计算机分析,VH和VL基因均为新发现的基因序列,符合功能性重排的鼠抗体可燮区基因特征,2E3－ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ（B）和轻链kⅢ家簇;而1A8－ScFv的VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ（A）和轻链кⅥ家簇。     

Induction of light chain expression in a pre-B cell line by fusion to myeloma cells

Pre-B cells, the first cells in the B-lymphocyte differentiation pathway which express immunoglobulin, have recently been shown to express cytoplasmic mu heavy chain (H) but not light chain (L). If, as is believed, pre-B cells are the precursors of immature B lymphocytes, which express surface IgM, the differentiation of pre-B cells to immature B lymphocytes must be accompanied by the expression of light chains. In this case, it should be possible for the progeny of a single pre-B cell to express a variety of light chains in association with the same heavy chain. We have tested this hypothesis by hybridizing a pre-B cell line 18-81 expressing only cytoplasmic mu chains with variant myeloma cells which do not express light chains. Hybridization of B-lymphoma cells with myeloma cells usually produces a hybrid with the phenotype of the more differentiated parent. In this case, the fusion resulted in the induction of light chain expression from the 18-81 genes and we have been able to demonstrate that independent hybrids express different light chains, in accordance with the hypothesis that a pre-B cell committed to expression of a single mu heavy chain can generate progeny expressing different slight chains.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.