鼠源单抗3G1单链抗体基因的构建及序列分析

利用Rr—PCR方法，从分泌抗汉滩病毒mAb的杂交瘤细胞系3Gl中扩增出抗体VH和VL基因，通过PCR方法获得1inker—VL基因，进而利用加端PCR技术，在扩增出的单抗体VH和1inker—VL基因两端加上限制性酶切位点，分别克隆入pUCl8中并测序．3Gl VH基因长360bp，编码120个氨基酸；VL基因长324bp，编码108个氨基酸．在pUCl8载体中将VH和1inker—VL基因连接成ScFv基因．     

鼠源抗AFB1噬菌体单链抗体库的构建与鉴定

研究以AFB1-BSA免疫的小鼠脾脏细胞为试验材料,利用RT-PCR技术,克隆了抗AFB1抗体重链和轻链可变区基因VH和VL,利用连接肽(Gly4Ser)3将VH和VL链接成单链抗体基因scFv,通过噬菌体展示载体pCANTAB-5E携带将其电转化E.coli TG1,经氨苄青霉素平板筛选,构建了库容为2.13×109 cfu/μg DNA的噬菌体单链抗体库,抗体库的克隆阳性率达到100%,且多样性良好,为高活性抗AFB1单链抗体的筛选提供了材料基础。     

抗α毒素单链抗体基因表达载体的构建

将2种抗A型产气英膜梭菌α毒素单链抗体（ScFv）基因ScFv－2E3和ScFV-1A8分别克隆至表达质粒pUC119,pET-20b,pET-28a和pHOG21中,构建了重组质粒PUC2E3和pUC-1A8,pET20b-2E3和pET20b-1A9,pET28a-2E3和pET28a-1A8以及pHOG-2E3和pHOG-1A8,然后分别转化至大肠杆菌中,提取质粒,并进行酶切鉴定和核苷酸序列分析,结果表明构建的重组质粒中均含目的ScFv基因片段,说明已成功构建了8个含ScFv基因的表达质粒。     

EL-4荷瘤鼠单链噬菌体抗体库的构建

目的:构建一个EL-4荷瘤鼠的单链噬菌体抗体库,为筛选高特异性和高亲和力的单链抗体做准备.方法:于C57小鼠腋区接种EL-4细胞,待肿瘤长大后,从脾细胞中提取总RNA,RT-PCR技术扩增小鼠抗体重、轻链可变区基因(VH、VL),用Linker奖VH和VL基因连成单链抗体可变区片段(scFv).双酶切后(Not Ⅰ、Sfi Ⅰ)与预备好的pCANTAB5E噬粒载体连接,转化入感受态TG1,构建EL-4荷瘤鼠的单链噬茵体抗体库.随机抽取转化后的20个克隆,用以检测外源DNA的转入情况.结果:PCR扩增出的VH约有340bp和VL约有320bp,scFv的长度约有750bp.转化后的TG1约有1.67×107个茵落,随机挑取20个克隆,双酶切显示五分之一的茵落转化了外源DNA片段,有效库容为3.34×106.结论:成功构建了EL-4荷瘤鼠的单链噬茵体抗体库.     

抗α毒素单链抗体基因表达载体的构建

将 2种抗A型产气荚膜梭菌α毒素单链抗体 (ScFv)基因ScFv - 2E3和ScFV - 1A8分别克隆至表达质粒pUC119,pET - 2 0b ,pET - 2 8a和pHOG2 1中 ,构建了重组质粒PUC -2E3和pUC - 1A8,pET2 0b - 2E3和pET2 0b - 1A8,pET2 8a - 2E3和pET2 8a - 1A8以及pHOG - 2E3和pHOG - 1A8,然后分别转化至大肠杆菌中 ,提取质粒 ,并进行酶切鉴定和核苷酸序列分析 ,结果表明构建的重组质粒中均含目的ScFv基因片段 ,说明已成功构建了 8个含ScFv基因的表达质粒     

丙溴磷人源ScFv抗体的筛选与鉴定

采用菌噬体表面展示技术,以丙溴磷（profenofos）为固相包被抗原,从半合成的菌噬体单链可变区抗体库中经过3轮“吸附-洗脱-扩增”筛选过程,获得抗原特异性和结合性较强的丙溴磷人源单链可变区抗体（ScFv）片段,片段为780 bp,免疫学检测结果表明阳性克隆株具有特异结合丙溴磷抗原的生物学活性.并对6株编码ScFv的序列进行了基因组序列测定及BLA ST分析,结果表明该抗体基因序列VH属于IGHV 1、VL属于IGLV 3,证明是新的抗体基因序列.     

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PAF单链抗体的基因构建及蛋白质3D模建

构建抗PAF单链抗体的基因并通过计算机模建分析几种不同连接类型的单链抗体空间结构差异。采用重叠延伸PCR方法扩增了VH和VL基因，形成抗PAF的VH-Linker-VL(ScFv)基因产物，并经菌落PCR和测序分析鉴定，通过Internet对其3D结构进行了模建与分析，研究结果表明：PCR产物电泳可见一条与目的基因大小一致的片段，经菌落PCR和测序分析鉴定，抗PAF单链抗体的基因构建成功；VH和VL在单链抗体中的顺序对单链抗体3D的空间结构有影响。     

抗弓形虫噬菌体单链抗体库的构建及筛选

采用弓形虫可溶性抗原攻击的小鼠,取小鼠脾脏从中提取细胞总RNA,通过RT-PCR扩增鼠源抗体VH和VL基因,并采用重叠PCR (SOE-PCR)方法构建ScFv基因,将其克隆入噬粒载体pCANTAB5E中,转化于感受态大肠杆菌TG1,通过辅助噬菌体M13K07援救构建噬菌体单链抗体库.从20个噬菌体克隆中筛选到15个具...     

Lack of heterogeneity in antihapten antibodies of a phylogenetically primitive shark

Individual mammals have the capacity to express at least one million distinct antigen binding specificities, implying a high degree of structural heterogeneity in the variable heavy and light chain (VH and VL) portions of the antibody molecules. Studies of higher vertebrate species suggest that this heterogeneity is created both through a sizeable repertoire of germ-line VH and VL genes and through random rearrangements of V and joining genes. Additional somatic mechanisms probably also contribute to the ultimate heterogeneity; one-third of murine plasmacytomas producing lambda 1 immunoglobulin carry a somatically mutated Ig1-V gene. The relative contributions of these various mechanisms to the overall imunoglobulin variability are difficult to evaluate. The production of different antibodies to a defined determinant in different individuals of an inbred mouse strain [for example, (3-iodo-4-hydroxy-5-nitrophenyl) acetyl (NIP) in CBA mice] suggests the involvement of somatic mutations or rearrangement but does not rule out the possibility that each individual CBA mouse expresses only a small random fraction from a large germ-line repertoire of V genes determining different anti-NIP binding sites. The opposite finding, that different individuals produce nearly identical antibodies to a defined determinant, would suggest the presence and expression of a limited number of germ-line genes without somatic alterations. Data presented here suggest that primitive sharks (Heterodontus fransisci) produce such antibodies to the hapten furyloxazolone.     

抗CPAα毒素单链抗体基因的克隆和表达及其生物学特性研究

应用RT—PCR技术 ,从分泌具有中和活性的抗A型产气荚膜梭菌 (CPA)α毒素单克隆抗体的杂交瘤细胞中 ,扩增出单链抗体 (ScFv)基因 ,并将其定向克隆于表达载体 pHOG2 1中 ,构建重组表达载体 pHOG 2E3,转化至大肠杆菌XL1 Blue中 ,筛选出表达菌株XL1 Blue(pHOG 2E3)。SDS PAGE分析结果表明 ,在 2 0℃用IPTG诱导培养时 ,表达的ScFv蛋白占菌体总蛋白的 2 5 %,ScFv蛋白主要以包涵体的形式存在 ,但在胞周质和培养上清中也能检测到ScFv蛋白 ,其中在胞周质中表达的ScFv蛋白占菌体可溶性蛋白的 4 %。生物学试验结果表明 ,ScFv基因表达产物不仅能够中和α毒素的磷酯酶C活性 ,而且对攻击致死剂量α毒素的小鼠产生良好的被动保护作用     

Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.     

Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.