Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

In-cell PCR from mRNA: amplifying and linking the rearranged immunoglobulin heavy and light chain V-genes within single cells.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.     

Direct cDNA cloning of the rearranged immunoglobulin variable region

A major problem in the study of multigene families is the effort required to clone and sequence these genes. We describe a method to rapidly clone and sequence immunoglobulin variable region gene sequences without constructing cDNA libraries. Because immunoglobulin variable-region genes are flanked by conserved sequences, we have been able to apply the polymerase chain reaction (PCR) to clone and sequence both the light- and heavy-chain rearranged immunoglobulin genes from small numbers of hybridoma cells. This method will greatly facilitate the construction of chimeric mouse/human monoclonal antibodies for immunoglobulin structural studies as well as for therapeutic use.     

Cloning and characterization of mouse growth hormone-releasing hormone (GRH) complementary DNA: increased GRH messenger RNA levels in the growth hormone-deficient lit/lit mouse

We have isolated and cloned the full length cDNA for mouse GH-releasing hormone (mGRH) from mouse hypothalamus using a recently described strategy involving the polymerase chain reaction technique (PCR). Degenerate oligonucleotide primers were selected based on short (six amino acids) conserved regions in the human and rat GRH peptides that would recognize DNA sequences encoding similar amino acids regardless of codon usage. Primer-extended cDNA was amplified by PCR on cDNA templates prepared by reverse transcribing total mouse hypothalamic RNA. After cloning and sequencing the initial product, the 3' and 5' ends of mGRH were generated using a separate PCR strategy (RACE protocol). The mGRH cDNA encodes a 103-amino acid reading frame, structurally similar to the human and rat GRH genes, containing a signal sequence, a 42-residue GRH peptide, and a 31-residue C-terminal region. Although the structures of mouse and rat GRH are highly conserved in the signal peptide and C-terminal region, there is considerable diversity in the GRH region, which exhibits nearly comparable homology with the rat (68%) and human (62%) structures. Differences between mouse and rat GRH were also found in the amino acid cleavage sites at the 5' and 3' ends of the mature peptide and at the polyadenylation signal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rapid cloning of rearranged immunoglobulin genes from human hybridoma cells using mixed primers and the polymerase chain reaction

A general method to directly obtain the DNA sequence of the variable regions of any immunoglobulin chain using a mixture of oligomer primers and the polymerase chain reaction (PCR) is described. Mixed oligonucleotide primers corresponding to the 5' signal peptide and a conserved 3' constant region primer were used for enzymatic amplification of each of the heavy and light chain variable regions of a human hybridoma producing a monoclonal antibody recognizing an epitope of gp120 of the human immunodeficiency virus 1. The amplified DNA segments were cloned and the sequence was determined for the heavy chain variable region. This method will greatly facilitate structural and functional studies of immunoglobulins by reducing the effort to clone and sequence the members of the immunoglobulin as well as other multigene families.     

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.     

Rapid verification of lambda-cDNA clones using mixed-base oligonucleotide as screening probe and sequencing primer

A rapid procedure has been developed for the isolation and verification of cDNA clones isolated from a cDNA library based on lambda vectors. Using information of the partial amino acid sequence of a protein, synthetic mixed-base oligonucleotides are first employed as a screening probe using the plaque hybridization procedure. The cDNA inserts of the clones obtained are then directly amplified by polymerase chain reaction (PCR) using primers flanking the cloning site of the vector. Besides being used for cloning into a plasmid vector, the amplified DNA's are also subjected to nucleotide sequence analysis using the same mixed-base oligonucleotides as sequencing primers. This approach allows sequencing through the region of the known amino acid sequence for direct verification of the authenticity of the clones obtained. This procedure has successfully been used for cloning and partial characterization of the gene coding for a platelet aggregation inhibitor.     

甲型H1N1流感病毒血凝素单克隆抗体轻链和重链可变区基因的巢式PCR扩增及序列分析

目的：采用巢式PCR对甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链基因进行扩增,对获得的基因进行序列分析,并找出克隆鼠Igκ轻链和重链可变区基因的通用方法。方法：设计22对扩增鼠Igκ轻链可变区和重链可变区基因的引物,对6株鼠抗人甲型H1N1流感病毒血凝素单克隆抗体的轻链和重链可变区基因进行克隆并测序,与NCBI公布的鼠免疫球蛋白序列比对分析。结果：巢式PCR方法可以有效避免单克隆抗体克隆过程的假基因,并且得到的单克隆抗体的氨基酸序列均符合鼠免疫球蛋白可变区特征。结论：建立了克隆鼠免疫球蛋白轻链和重链可变区基因的通用方法,为后期克隆鼠源性单克隆抗体的可变区基因提供了基础,并为研究甲型H1N1流感病毒血凝素与抗体的结合位点提供了实验数据。     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

Cloning and characterization of cDNAs coding for heavy and light chains of a monoclonal antibody specific for pre-S2 antigen of hepatitis B virus.

Binding specificity of a monoclonal antibody (mAb) (kappa, gamma 2b) H8 which can react with the pre-S2 peptide of hepatitis B virus (HBV) was determined by Western blot analyses. From the hybridoma cell line secreting mAb H8, poly(A)+ RNA was prepared and used as a template for cDNA synthesis and cloning. Full-length cDNAs coding for the heavy and kappa light chains of the mAb were cloned from the cDNA library and characterized by nucleotide (nt) sequence analyses and N-terminal amino acid sequencing. The sequence analyses revealed that both heavy and light chain-specific cDNAs are functional, and the variable regions of the heavy and light chains are members of mouse heavy chain subgroup III(c) and light chain group I, respectively. Comparison of the nt sequences with mouse immunoglobulin genes listed in the GenBank data base show that the cDNAs have not been previously reported. The cDNAs will be used for the construction of a therapeutic antibody for HBV infection.     

抗人AFP抗体噬菌体呈现文库的构建筛选及基因序列测定

从人ＡＦＰ免疫小鼠脾细胞ｍＲＮＡ中扩增出全套抗体Ｖ区基因并随机拼接为ＳｃＦｖ基因,构建全套ＳｃＦｖ基因噬菌体呈现文库,经两轮ｐａｎｎｉｎｇ筛选富集,一次从９４个单个重组噬菌体克隆中筛选到１４个具有ＡＦＰ结合活性的克隆,测定两个阳性克隆中ＳｃＦｖ基因的核苷酸序列,获得一个Ｖ＿Ｈ基因和两个Ｖ＿Ｋ基因,其基因序列分别与鼠ＩｇＶ＿ＨＪ５５８、Ｖ＿ｋ－ＯＸ１和Ｖ＿ｋ４／５家族同源性最高;推导出的氨基酸序列中均含有抗体Ｖ区特征性的两个恒定的半胱氨酸残基、具有明确的三个ＣＤＲ和四个ＦＲ序列,表明这三个基因均系新发现的功能性鼠抗体Ｖ区基因序列。     

鼠源抗B型肉毒毒素单链抗体噬菌体文库的构建筛选及抗体免疫学活性的初步研究

B型肉毒毒素重链C-端片段(BoNTB/Hc)经金属螯和层析法纯化后免疫Balb/c小鼠,从其脾淋巴细胞中提取总RNA,反转录成cDNA,用抗体可变区混合引物进行全套抗体重、轻链可变区基因的扩增,体外随机装配成单链抗体(scFv)。将其克隆至pCANTAB5E中,构建单链抗体噬菌体抗体库。结果表明经过4轮"吸附-洗脱-扩增"的富集过程,筛选获得高亲和力的克隆。序列测定符合抗体可变区结构特点。     

抗人小细胞肺癌单克隆抗体的重链变区基因的体外扩增、克隆和序列分析

本文从抗人小细胞肺癌单克隆抗体杂交瘤细胞中抽提总RNA,合成第一链cDNA,直接用PCR技术(polymerase chain reaction)扩增出351bp的重链变区基因(V_H),克隆至pUCV_(NP)-PCR载体上,经筛选得一批插入片段为351bp的阳性克隆,经核苷酸序列分析研究,证实已获得了该单克隆抗体的重链可变区基因。     

Heterozygous mutations at the mut locus in fibroblasts with mut0 methylmalonic acidemia identified by polymerase-chain-reaction cDNA cloning.

Genetic defects in the enzyme methylmalonyl CoA mutase cause a disorder of organic acid metabolism termed "mut methylmalonic acidemia." Various phenotypes of mut methylmalonic acidemia are distinguished by the presence (mut-) or absence (mut0) of residual enzyme activity. The recent cloning and sequencing of a cDNA for human methylmalonyl CoA mutase enables molecular characterization of mutations underlying mut phenotypes. We identified compound heterozygous mutations in a mut0 fibroblast cell (MAS) line by cloning the methylmalonyl CoA mutase cDNA by using the polymerase chain reaction (PCR), sequencing with internal primers, and confirming the pathogenicity of observed mutations by DNA-mediated gene transfer. Both mutations alter amino acids common to the normal human, mouse, and Propionibacterium shermanii enzymes. This analysis points to evolutionarily preserved determinants critical for enzyme structure or function. The application and limitation of cDNA cloning by PCR for the identification of mutations are discussed.     

Characterization and molecular cloning of neurotoxic phospholipases A2 from Taiwan viper (Vipera russelli formosensis).

Two phospholipases A2 (PLA2s), designated as RV-4 and RV-7 were purified from venom of the Taiwan Russell's viper (Vipera russelli formosensis) by gel-filtration and reverse-phase HPLC. Their primary structures were solved by both protein sequencing and cDNA cloning and sequencing. The cDNA synthesized was amplified by the polymerase-chain reaction using a pair of synthetic oligonucleotide primers corresponding to the N- and the C-terminal flanking regions of the enzymes. The deduced amino acid sequences of RV-4 and RV-7 were 92% identical to those of the vipoxin and vipoxin inhibitor, respectively, from the Bulgarian Vipera a. ammodytes. RV-4 itself was neurotoxic, whereas RV-7 had much lower enzymatic activity and was not toxic. The low enzymatic activity of RV-7 may be attributed to five acidic residues at positions 7, 17, 59, 114 and 119, which presumably impair its binding to aggregated lipid substrates. Based on the sequence comparison among all the known group II PLA2s, residues 6, 12, 76-81, and 119-125 were identified as important for the neurotoxicity. RV-4 and RV-7 exist in the crude venom as heterodimers, which were again formed by mixing together the HPLC-purified RV-4 and RV-7. Moreover, RV-7 inhibited the enzymatic activity of RV-4 in vitro but potentiated its lethal potency and neurotoxicity. It is suggested that RV-7 may facilitate the specific binding of RV-4 to its presynaptic binding sites, probably by preventing its non-specific adsorption.     

Patterns of variation in MHC class II beta loci of the little greenbul (Andropadus virens) with comments on MHC evolution in birds

We have isolated major histocompatibility complex (MHC) class II beta loci from the little greenbul (Andropadus virens), an African songbird. We utilized preexisting information about conserved regions of the avian MHC to design primers to amplify a pool of sequences representing multiple loci. From this pool, a unique locus spanning 1109 bp that we designate as Anvi-DAB1 was cloned and sequenced. We designed locus-specific primers based on this sequence information and amplified six alleles from seven individuals. Compared to other A. virens MHC sequences obtained from genomic DNA or cDNA, the variability of sequences from Anvi-DAB1 was low and the ratio of nonsynonymous to synonymous substitution was much less than one, suggesting that Anvi-DAB1 may either be a pseudogene or a nonclassical MHC locus. Phylogenetic analysis revealed that the Anvi-DAB1 locus was highly divergent when compared with other passerine or A. virens genomic or transcribed MHC sequences. The use of conserved MHC primers followed by analysis of cloned sequences allows rapid isolation of MHC loci from exotic species and avoids laborious large-scale cloning and sequencing.     

Expression in COS cells of a mouse-human chimaeric B72.3 antibody

B72.3 is a mouse hybridoma cell-line secreting an IgG1 antibody which recognises an epitope on a tumour-associated antigen, TAG-72. This high molecular weight mucin-like molecule is found on a variety of human neoplasms, including colon, breast and ovarian carcinomas. Chimaeric immunoglobulin genes with the B72.3 specificity have been constructed by joining the mouse variable regions from cDNA clones to human genomic constant regions using recombinant DNA techniques. The chimaeric heavy and light chain immunoglobulin genes were placed under the control of a strong viral promoter, and co-transfected into COS-1 cells. SDS-PAGE analysis of the 35S-labelled products demonstrated that the transiently expressed antibodies were correctly synthesised and assembled. The specific binding characteristics of the parent B72.3 antibody were retained by the chimaeric antibody in an antigen-based ELISA. This system gave sufficiently high transient expression of the chimaeric antibody molecules to allow rapid physical and immunological characterisation of the engineered gene products.     

Expression of mouse::human immunoglobulin heavy-chain cDNA in lymphoid cells

A chimeric mouse variable::human constant immunoglobulin heavy-chain gene was expressed in transfected mouse Sp2/0 cells. The chimeric immunoglobulin genes were integrated in tandem in the genome of stably transformed cells. These integrated gene copies were amplified by selection with a second drug marker. The gene amplification led to an increase in the expression of chimeric heavy-chain protein. The level of gene expression appears to be related to the site of integration; a few gene copies in one transfectant can yield as much heavy-chain protein as many copies in a second transfectant. In addition, we found that an adventitious oligo(C) sequence, introduced by our method of gene construction at a site located 8 nt residues downstream from a splice acceptor, can apparently direct splicing towards a cryptic splice acceptor downstream from the oligo(C).