Cloning of a polymorphic DNA fragment from the genome of Leishmania donovani

Recombinant DNA clones, containing highly repetitive DNA sequences, have been isolated from a Leishmania donovani genomic DNA library prepared in the replacement vector lambda gt.WES.lambda B. Two clones, probably telomeric in location, have been characterised and show a restriction fragment size polymorphism. Evidence is presented which suggests that L. donovani is diploid for this cloned genomic locus.     

Cloning and expression of genomic DNA sequences coding for putative erythrocyte membrane-associated antigens of Plasmodium falciparum

Genomic DNA fragments of Plasmodium falciparum generated by mung bean nuclease digestion were cloned in the lambda expression vector lambda JK2. The resulting library was screened with a rabbit antiserum raised against purified membranes of P. falciparum-infected erythrocytes and with a serum pool from immune humans from an endemic area of Liberia. Positive clones were rescreened with a series of human and monkey sera. Twelve selected clones were analysed in detail. Four of them corresponded to already described membrane-associated P. falciparum antigens. The other positive clones contained inserts which, according to the nucleotide sequence, Southern blot analysis and immunological characteristics, correspond to so far unknown antigens.     

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.     

Cloning and expression of the streptococcal C5a peptidase gene in Escherichia coli: linkage to the type 12 M protein gene.

A genomic library of Streptococcus pyogenes CS24 DNA was constructed by cloning streptococcal DNA partially digested with Sau3A into the lambda replacement vector EMBL3. The expression of streptococcal C5a peptidase (SCP) was analyzed by radioimmunoassay with hyperimmune rabbit serum. Two clones, lambda 4.1 and lambda 4.2, were found to express the desired antigen, and various DNA fragments from the hybrid bacteriophage lambda 4.1 were subcloned into the plasmid vector pUC9 in Escherichia coli. One of the recombinant plasmids, designated pTT1, contained a 5.8-kilobase (kb) streptococcal DNA insert. Analysis of total cellular protein from this E. coli clone by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blotting identified a 140,000-Mr protein, similar in size to the native protein purified from S. pyogenes. Cloned SCP was functionally active, as shown by its ability to inhibit C5a-mediated chemotaxis. By deletion analysis with both restriction endonucleases and BAL 31 nuclease, the SCP gene was localized to a 4.3-kb segment of DNA. Southern hybridization experiments showed that the type 12 M protein-coding sequence is also present in the hybrid phage lambda 4.1, at approximately 2 kb upstream of the SCP structural gene. Western blot analysis indicated that the cloned streptococcal DNA in lambda 4.1 directed the expression of both SCP and M12 protein.     

Beta tubulin gene of the parasitic protozoan Leishmania mexicana

A genomic DNA library was generated with Sau3A cut DNA derived from promastigotes of Leishmania mexicana amazonensis and the lambda vector EMBL3. The library was screened for beta tubulin clones using 32P-labeled heterologous probe of chicken beta tubulin cDNA. From the various genomic clones the one designated 23.1, which gave the simplest hybridization banding pattern, was further characterized. The leishmanial insert DNA was subcloned into plasmid vectors and the resulting clones were designated as T11, T28 and T50. Using these clones leishmanial beta tubulin coding region was sequenced by the dideoxy method. The result shows that the beta tubulin has 445 amino acids, a carboxyl terminal tyrosine, and no intron. Leishmanial beta tubulin has 93% amino acid sequence similarity with that of trypanosome and 82% with that of man: and there is a strong bias in codon usage for codons possessing guanine or cytosine in the third base.     

Characterization of baculovirus p10 synthesis using monoclonal antibodies

A series of monoclonal antibodies were produced against virion proteins of the multicapsid nuclear polyhedrosis virus of Orgyia pseudotsugata (OpMNPV). Four of these antibodies reacted with a protein of 14 kd on Western blots of electrophoretically separated OpMNPV virion proteins. These antibodies were used to identify immunoreactive clones from a lambda gt11 expression library of OpMNPV DNA. By hybridization of insert DNA from the lambda gt11 clones to blots of digests of OpMNPV genomic DNA, and by sequencing the ends of the lambda gt11 inserts, these clones were shown to contain a portion of the p10 gene. The regions containing epitopes recognized by the four monoclonal antibodies were located using fusion proteins made from selected portions of the p10 reading frame in a trpE vector. One of the p10 antibodies was used to characterize p10 synthesis in infected Lymantria dispar cells by using Western blots and immunofluorescent staining. The p10 protein was detected with immunofluorescent microscopy at 14 hr postinfection and by 20 hr it formed intensely staining cytoplasmic structures. On Western blots of infected cells, two forms of p10 (of about 14 and 15 kd) were observed. One of the p10 monoclonal antibodies showed a strong cross-reaction with cytoskeletal structures in uninfected insect cells and rat fibroblasts.     

Isolation of a gene segment expressed by mature schizonts of Plasmodium knowlesi

Fragments of Plasmodium knowlesi DNA, generated by mung bean nuclease digestion, were ligated into the lambda gt11 vector. This expression library was immunoscreened with a serum which inhibits invasion of erythrocytes by merozoites in vitro and whose primary specificity is directed against a Mr 140 000 merozoite surface antigen. One of the isolated clones contained a 125 base pair insert which hybridized to a 1.8 kilobase species of schizont RNA, indicating that this insert is part of a gene expressed during schizogony.     

人源性抗地高辛单链抗体(scFv)及diabody的获取

目的:从大容量噬菌体抗体库中筛选人源性抗地高辛单链抗体(scFv),并构建双价抗体(diabody)。方法:以固相化的Dig对构建的大容量噬菌体抗体库进行筛选,4轮后挑取集落,用ELISA法鉴定其特异性,并对抗Dig阳性抗体基因进行DNA指纹分析及测序分析。选取活性好的抗体基因进行改造,构建diabody。结果:在抗体库的筛选过程中可见到明显的富集现象,获得4株可与Dig特异性结合的人源单链抗体,经DNA指纹分析及测序分析证明为不同抗体基因,分泌抗Dig抗体的阳性克隆的可变区基因轻链均属于λ第1亚群,重链基因分别属于第3和第4亚群。选取4号克隆进行基因改造,构建diabody的表达载体,获得活性较好的diabody。结论:利用噬菌体抗体技术获得了人源性抗Dig的抗体,并改造成应用前景较好的diabody,为临床诊断及治疗洋地黄类药物的中毒提供了具有实用价值的人源抗体。     

Expression of Mycoplasma pneumoniae antigens in Escherichia coli.

A genomic library of Mycoplasma pneumoniae was generated by using bacteriophage lambda EMBL3 as the vector. Screening of the library for the expression of M. pneumoniae protein antigens with adsorbed anti-M. pneumoniae serum revealed strong reactivity from a third of those clones which contained mycoplasma DNA inserts. Three of the most highly reactive clones were analyzed in detail and found to synthesize discrete mycoplasma proteins. Two carried overlapping fragments of mycoplasma DNA which encoded a protein that was readily detected in Escherichia coli after infection with recombinant bacteriophage. The third clone contained a novel mycoplasma DNA fragment which directed the synthesis of two additional mycoplasma proteins. Further screening of the library with antiserum raised against the major M. pneumoniae adhesin protein P1 (165 kilodaltons [kDa]) yielded one clone which produced an immunologically reactive protein of 140 kDa. Adsorption of anti-P1 serum by this clone selected a population of antibodies that were reactive with M. pneumoniae adhesin P1 (165 kDa). These results demonstrate that immunologically active M. pneumoniae proteins are synthesized in E. coli.     

Procaryotic expression of phosphorylated tegument protein pp65 of human cytomegalovirus and application of recombinant peptides for immunoblot analyses.

The tegument of human cytomegalovirus (HCMV) contains a phosphorylated protein of 65 kilodaltons, termed pp65, which was reported to carry significant epitopes for the stimulation of the humoral immune response during natural infection. A monoclonal antibody directed against this protein was used to screen a lambda gt11 cDNA library for recombinant polypeptides. Two DNA fragments from purified lambda clones and one fragment from genomic DNA were used for cloning in a bacterial high-level expression vector. The resulting fusion proteins were tested for their reactivity with a panel of monoclonal antibodies directed against pp65 and with polyspecific anti-HCMV rabbit antisera. The binding site for all the monoclonal antibodies tested was found to be contained in one of the recombinant proteins with a viral portion of 26 amino acids. Immunoblot analyses with HCMV-positive human sera revealed that pp65 alone is not a reliable antigen for serodiagnosis but may be very useful in combination with other HCMV proteins.     

Structure of extrachromosomal circular DNAs generated by immunoglobulin light chain gene rearrangements

Recombination at the immunoglobulin kappa or lambda light chain locus generates extrachromosomal circular DNAs. We have isolated circular DNAs from adult mouse spleen cells and prepared a circular DNA clone library. We characterized four J kappa-positive and one J lambda 1-positive clones. The J kappa-clones contained both coding and signal joints of V kappa-J kappa joining, and the J lambda 1-clone contained a signal joint of V lambda 1-J lambda 1 joining. Genomic organization of the V kappa gene families used in these joints suggested the excision of circular DNA preceded by inversion. A specific dinucleotide (P) insertion in the coding joint was observed in two clones. Three coding joints were out of frame and one clone had an in-frame coding joint, although possibly combined with a pseudo-V kappa gene. These kappa-positive circular DNAs are possibly excised from the chromosome by secondary recombinations which replace non-productive primary rearrangements.     

Entamoeba histolytica: cloning and characterization of actin cDNA

In order to study gene expression in the human parasite Entamoeba histolytica, a cDNA library of E. histolytica strain 200:NIH was constructed using the phage vector lambda gt10. Three cDNA clones (A, B and C) were selected for further analysis. Each of the three clones hybridized to a distinct mRNA. Two of these mRNAs were translated in vitro after hybrid selection, and yielded distinct translation products. One of these mRNAs, selected by hybridization to clone A, encodes the most abundantly expressed protein in E. histolytica. DNA sequence analysis of this cDNA clone identified the DNA as that encoding actin. The deduced amino acid sequence of E. histolytica actin resembles both cytoplasmic and muscle actins and has an unusual N-terminal glycine residue. We have shown that a family of actin genes is present in E. histolytica. Six different E. histolytica actin clones were obtained from a lambda gt10 genomic library using subcloned cDNA probes. Southern analysis of three different E. histolytica strains (200:NIH, Rhaman, and HM-1:IMSS) revealed at least four different actin genes. Strain HM-1:IMSS, however, differs by the presence of an additional actin gene.     

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.     

Plaque antibody selection: rapid immunological analysis of a large number of recombinant phage clones positive to sera raised against Plasmodium falciparum antigens

A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.     

Cloning of Mycobacterium bovis BCG DNA and expression of antigens in Escherichia coli.

A gene bank of Mycobacterium bovis BCG DNA in Escherichia coli was constructed by cloning Sau3A-cleaved mycobacterium DNA fragments into the lambda vector EMBL3. The expression of mycobacterial antigens was analyzed by Western blotting with hyperimmune rabbit sera. Among 770 clones tested, several were found that produced various mycobacterial antigens in low amounts, with concentrations generally close to the detection limit. One particular clone was chosen for further investigation. This clone produced a 64-kilodalton (kDa) antigen. By placing the lambda promoter PL in front of the structural gene of this antigen, an overproducing E. coli strain was obtained. Rocket-line immunoelectrophoresis experiments showed that antigens cross-reacting with the 64-kDa protein are present in a wide variety of mycobacteria and also in so-called purified protein derivatives which are routinely used for skin tests. Preliminary experiments indicate the presence of antibodies against the 64-kDa antigen in sera from tuberculosis patients.     

Molecular cloning of DNA coding for outer membrane proteins of Haemophilus influenzae type b.

DNA from Haemophilus influenzae type b was cloned in Escherichia coli with a vector lambda gt11 Amp1. Clones producing antigens reactive with hyperimmune rabbit antisera were identified by colony radioimmune assay. A second screening with hyperimmune serum adsorbed with intact H. influenzae type b bacteria was used to identify those clones producing surface-exposed outer membrane proteins. The proteins expressed in E. coli were coupled to Sepharose and used to affinity purify antibodies which were tested for reactivity with outer membrane vesicles. It was found by Western blotting that the clones were producing antigens corresponding to Mr 49,000, 39,500, and 35,000 major outer membrane protein or antigens of H. influenzae type b. Additional clones could be detected by human serum, but their reactivity was not removed when serum was adsorbed with intact bacteria. One of these studied in more detail was found to produce an antigen present in H. influenzae type b lysate but not in outer membrane vesicle preparations.     

Structure of viral DNA in a rat cell line, GY1, transformed by Ad12 HindIII fragment-G

The cell line GY1, established by transformation of a rat cell line 3Y1 with the Ad12 HindIII fragment-G (leftmost 6.8%, nucleotide 1 to 2322), contains more than 100 viral copies per haploid genome. The viral DNAs in this cell line were cloned into a phage vector, lambda gtWES lambda B, and recloned into pBR322 with their flanking cellular DNAs. Independently isolated 39 clones were analyzed by restriction enzyme cleavage and Southern blot hybridization experiment and divided into 11 classes. Some of classes contained multiple identical clones, at maximum 16 clones. It may be interpreted that amplification of some of the recombined sequences had occurred after the multiple integrations of transfected DNAs within cells. Using five clones from different classes the sequences of recombination sites were determined. Viral DNAs deleted with varying degrees at both ends were flanked by quite different cellular sequences in different clones and no common sequences were revealed around viral-cellular junctions. Tandemly repeated viral DNAs were found in one of the clones to be integrated in a head to tail manner into cellular DNA. The linkage of these two viral DNAs had occurred at the site where parental viral DNAs shared 2 bp. Palindrome structures could be constructed around viral-cellular and viral-viral junction sites and around the regions of parental viral DNAs corresponding to the junction sites in all of the cases investigated.     

Biological and immunological characterization of a cloned cholera toxin-like enterotoxin from Salmonella typhimurium

A chromosomal DNA fragment, encoding an enterotoxin gene of Salmonella typhimurium Q1, was cloned into bacteriophage EMBL3 and plasmid vector pBR322. The recombinant clones lambda B8 and pC1 were identified using a synthetic oligonucleotide probe made to the B subunit region of the cholera toxin gene (ctx). Cell lysates of Escherichia coli VCS257 [lambda B8] induced fluid secretion in rabbit intestinal loops, while lysates of E. coli DH5 alpha [pC1] failed to elicit an enterotoxic response in this model. Both lysates and partially purified preparations elongated Chinese hamster ovary (CHO) cells, elevated cellular cAMP and PGE2, and bound to ganglioside GM1. The biological activity associated with the cloned enterotoxin was neutralized by monospecific antiserum to cholera toxin (CT). Immunoblots of pC1 and lambda B8 lysates probed with anti-CT, exhibited a 30 kDa protein similar to that of pJM17, which carried the ctx gene. Under non-dissociating conditions, anti-CT immunoblots of the same lysates revealed two proteins, one corresponding in size to the holotoxin and the other to CT-A. When analysed by DNA-directed protein synthesis in vitro, both pC1 and lambda B8 DNA expressed two unique proteins (30 and 11 kDa) similar to that of pJM17.     

Isolation of lambda amp3 genomic recombinants coding for antigens of Eimeria tenella

Eimeria are the causative agents of coccidosis, a disease which is of world wide economic importance in the poultry industry. Immunity resulting from infection is species-specific and both antibody and cell-mediated responses have been implicated. As an initial step in the development of a genetically-engineered vaccine against coccidiosis, libraries of EcoRI-digested genomic DNA from E. tenella have been constructed in Escherichia coli using the expression vector lambda amp3. Screening of the libraries with serum from chickens immunized by infection has identified at least 24 different recombinant phage which produce eimeria antigens fused to beta-galactosidase. A significant proportion of the Eimeria DNA inserts cross-hybridise with each other and contain sequences which are highly represented in the genome. The identification of these clones will enable the isolation of intact genes from E. tenella DNA and facilitate detailed analysis of the antigens and immune responses.