抗小细胞肺癌单抗轻链变区基因的体外扩增、克隆及其部分顺序分析

本文通过设计一对分别位于鼠免疫球蛋白轻链变区基因(V_k)FR_1和FR_4骨架区的通用引物,利用PCR技术首次从抗小鼠肺癌单克隆抗体杂交瘤细胞株染色体组总DNA中直接进行体外扩增,扩增出的特异条带分子量在320～340bp之间,扩增产物经酶解克     

HLA—A2单抗轻链可变区基因的克隆及其序列分析

以ＨＬＡ－２单抗ｍＲＮＡ为模板，设计了３对特异性引物，采用ＰＣＲ技术，扩增和克隆出单以链可变区基因并应用双脱氧链式终止法及计算机基因文库测定和分析了其氨基酸和核苷酸序列。结果证明，本组单抗轻链由不同的基因家族编码。     

抗CD3单克隆抗体重、轻链可变区基因的克隆及序列分析

目的：抗CD3单克隆抗体（WuT3)可变区基因克隆及序列分析。方法；采用RT-PCR技术，从WuT3杂交瘤细胞总RNA中扩增VH，VL片段，经酶切后通过链接反应构建重组克隆载体，测序鉴定。结果：通过国际联机检索发现VH，VL基因与Ig同源，分别符合小鼠IgVH,Vк基因特征。VH基因属于鼠重链VH第ⅡB亚类，全长363bp，可编码121个氨基酸；VL基因属于鼠к轻链第Ⅲ亚类，全长330bp，可编码110个氨基酸，结论：成功获得WuT3单抗的重，轻链可变区基因。     

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...     

大鼠抗汉滩病毒单抗重链可变区基因的体外扩增、克隆和序列分析

大鼠抗汉滩病毒单抗重链可变区基因的体外扩增、克隆和序列分析程卫青，杨为松，徐海峰，白雪帆，尚高峰（第四军医大学唐都医院传染科西安７１００３２）刘健，郭虹汾，李斯德（上海市肿瘤研究所免疫室，上佝２０００３２）目前基因工程抗体的研究正方兴未艾。制备基因工...     

抗结肠癌单抗κ轻链基因的克隆和序列分析

小鼠抗结肠癌单抗ＣＬ－３经体内、外研究证实它对结肠癌细胞具有很高的特异性。为了使该单抗能够应用于肿瘤导向治疗，我们采用基因工程技术，从ＣＬ－３杂交瘤细胞系中克隆出了ＣＬ－３单抗κ轻链基因，并对该基因的全部核苷酸序列进行了测定。     

CEA单抗轻重链可变区基因的克隆和序列分析

从分泌具有高亲和力和特异性的小鼠抗癌胚抗原（ＣＥＡ）的单抗杂交瘤细胞株Ｃ５０中提取总ＲＮＡ，通过ＲＴＰＣＲ扩增并克隆了抗体的轻、重链可变区基因。核苷酸序列分析表明所克隆基因分别为抗体轻、重链可变区基因。     

人抗体κ轻链可变区基因的克隆及序列分析

人抗体κ轻链可变区基因的克隆及序列分析阎岩徐志凯姜绍谆王海涛尹文薛小平赵茜白文涛（第四军医大学微生物学教研室，西安７１００３２）用噬菌体抗体库技术制备人ＭｃＡｂ可以克服用常规方法难以克服的困难，如融合率及阳性率低、杂交瘤不稳定和抗体产量低等。扩增人...     

Cloning and sequencing of human lambda immunoglobulin genes by the polymerase chain reaction

Universal oligonucleotide primers, designed for amplifying and sequencing genes encoding the rearranged human lambda immunoglobulin variable region, were validated by amplification of the lambda light chain genes from four human heterohybridoma cell lines and in the generation of a cDNA library of human V lambda sequences from Epstein-Barr virus-transformed human peripheral blood lymphocytes. This technique allows rapid cloning and sequencing of human immunoglobulin genes, and has potential applications in the rescue of unstable human antibody-producing cell lines and in the production of human monoclonal antibodies.     

Cloning by polymerase chain reaction of a new mouse TGF-beta, mTGF-beta 3

Two TGF-beta s, TGF-beta 1 and 2, have previously been isolated from the mouse. Here we report the isolation of a murine TGF-beta 3 cDNA. RNAs extracted from 15-day-old mouse embryos and several mouse cell lines were reverse-transcribed. These cDNA mixtures were used as substrates for polymerase chain-reaction amplifications, using oligonucleotides designed on the basis of known human and chicken TGF-beta 3 sequences, including the initiation and stop codons. Several overlapping cDNAs containing either the amino-terminal domain or the carboxy-terminal domain, as well as the complete 1.2-kb coding region of the mouse TGF-beta 3 cDNA were obtained. The mouse TGF-beta 3 coding region is 1230 nucleotides long and codes for a 410 amino acid polypeptide very similar to its human counterpart. This cDNA hybridizes to a unique 3.5-kb RNA and is differentially expressed in various mouse tissues and at different embryonic stages.     

Oligonucleotide primers for polymerase chain reaction amplification of human immunoglobulin variable genes and design of family-specific oligonucleotide probes

In recent work, the polymerase chain reaction (PCR) has been used to amplify rearranged mouse and human immunoglobulin heavy and kappa light chain variable (V) genes. Here we have optimized the design of the PCR primers for human V genes and used them to amplify cDNA from human peripheral blood lymphocytes. Cloning and sequencing revealed a diverse repertoire of V genes, and the presence of members of each human V gene family. After alignment of the sequences, we identified a region conserved within V gene families, but differing between families, and used this to design family-specific oligonucleotide probes.     

Simultaneous sequencing of multiple polymerase chain reaction products and combined polymerase chain reaction with cycle sequencing in single reactions

DNA sequencing is considered the gold standard for nucleic acid identification and mutation detection. However, sequencing is labor intensive because it requires previous amplification and only a single sequence is analyzed at a time. We developed two novel strategies that substantially improve DNA sequencing. The first allows multiple polymerase chain reaction (PCR) products to be sequenced in a single sequencing reaction and analyzed simultaneously in a single lane or capillary. Simultaneous sequencing by this method, designated "SimulSeq," can provide either simultaneous single-direction sequencing of multiple genes or simultaneous forward and reverse sequencing from a single gene. In the second approach, designated "AmpliSeq," we demonstrate a technique combining PCR amplification and sequencing in a single reaction that is analyzed in a single lane or capillary. We demonstrate combined PCR with short bidirectional sequencing, and combined PCR with unidirectional sequencing. We anticipate that these methods will have utility in research and clinical settings where panels of mutations or large numbers of samples are be analyzed and/or when turnaround time is critical.     

Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction.

The isolation of the rearranged immunoglobulin genes from a hybridoma cell line, which is a prerequisite for the construction of a recombinant antibody, can easily be achieved by polymerase chain reaction. Here we demonstrate that this method, which was originally described for cloning murine immunoglobulin genes from cDNA, is also applicable for rat genes. We show that the procedure also works with crude cell lysates as starting material, thereby greatly reducing the time required for sample preparation. In addition we have sequenced the nonfunctional heavy chain variable gene of the fusion partner X63Ag8.653, which was readily amplified from our hybridoma cells, and whose sequence has been so far unknown.     

The absence of DNA polymerase kappa does not affect somatic hypermutation of the mouse immunoglobulin heavy chain gene

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase kappa (Polkappa), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polkappa, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polkappa is involved in SHM, we immunized Polkappa-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polkappa knockout mice and control mice. These results suggested that Polkappa is not essential for this process.     

Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction

We have optimized primers for cloning libraries of murine heavy and light chain variable regions using the polymerase chain reaction. Since we are interested in cloning murine Fab fragments for expression in bacterial cells, the heavy chain primers were designed to clone Fd fragments comprising the heavy chain variable domain and the first domain of the IgG constant region. The light chain primers were designed to clone the entire murine kappa chain. Using ten degenerate 5′ primers and a degenerate 3′ primer to amplify murine Fd and seven degenerate 5′ primers with a single 3′ primer to amplify kappa chains, a diverse repertoire of mouse variable regions was cloned from mouse spleens.     

The variable genes and gene families of the mouse immunoglobulin kappa locus.

In this report 118 mouse Vkappa genes are described which, together with the 22 Vkappa genes reported previously (T. Kirschbaum et al., Eur. J. Immunol. 1998. 28: 1458-1466) amount to 140 genes that had been cloned and sequenced in our laboratory. For 73 of them cDNAs are known, i. e. they have to be considered functional genes, although 10 genes of this group have 1-bp deviations from the canonical promoter, splice site or heptanucleotide recombination signal sequences. Twenty Vkappa genes have been defined as only potentially functional since they do not contain any defect, but no cDNAs have been found (yet) for them. Of the 140 Vkappa genes 47 are pseudogenes. There are indications that two to five Vkappa genes or pseudogenes exist in the kappa locus which we have not yet been able to clone. The 140 Vkappa genes and pseudogenes were assigned to 18 gene families, 4 of them being one-member families. This differs from previous enumerations of the families only by the combination of the Vkappa9 and Vkappa10 families and by the addition of the Vkappa dv gene as a new separate family. Sequence identity usually was 80% or above within the gene families and 55-80% between genes of different families. Many of the mouse Vkappa gene families show significant homologies to the human ones, indicating that in evolution Vkappa gene diversification predated the divergence of the primate and rodent clades.     

Screening human antibody libraries against carcinoma cells by affinity purification and polymerase chain reaction

Bacterial scFv clones from a naïve antibody library have been isolated against cancer cell antigens with AffiSelect, a novel screening method that indirectly identifies candidate library members via an antigen reporter gene. The first step is the coating of carcinoma cell surface epitopes (antigen) with either mAbs, scFvs or phages (library members). Upon binding to a cell surface ligand, the library member generates a linking moiety. This facilitates magnetic affinity purification of the antibody–cancer cell complexes, detected by the polymerase chain reaction (PCR) using the β-actin gene of the cancer cell as the target. Combining these well-known methods resulted in a higher resolution than a comparable cell-based ELISA method of detection. We have isolated human scFv antibodies against surface antigens of a lung carcinoma cell line. These were identified from a polyclonal mixture of phage display-enriched library clones comparing PCR patterns of the carcinoma cell line with the two negative cell types, HUVEC and peripheral blood cells (PBLs). The positive clones were sequenced and verified by FACS.     

Detection of human papillomavirus DNA in cervical cancer tissue by the polymerase chain reaction

A method for detecting HPV DNA in cervical cancer tissue was developed without using isotopes. The DNA samples from the cancer tissues were first subjected to amplification by PCR, followed by polyacrylamide gel electrophoresis to identify the specific amplified fragment. The specificity and sensitivity of the PCR method are described. Compared with the dot hybridization technique, it is shown that the method is able to detect HPV DNA in cervical cancer tissues.