Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

Cloning and characterization of Taenia saginata paramyosin cDNA

A ZAP-express cDNA library of Taenia saginata metacestodes was constructed. Antibody screening yielded a clone with an insert of 3,408 bp, an open reading frame of 2,589 bp, a deduced sequence of 863 amino acid and a molecular mass of 98.89 kDa. Alignments of the predicted amino acid sequence showed identity with paramyosins from several species: 98.8% with Taenia solium, 96.3% with Echinococcus.granulosus and about 70% with Schistosoma spp. The insert was expressed and purified. A collagen binding assay was performed which showed that T. saginata GST-paramyosin retained this property in a dose-dependent manner. Problems were encountered due to high backgrounds in serological assays in the homologous T. saginata system. However, the recombinant paramyosin was recognized by antibodies present in 31.6% of sera from T. solium seropositive cysticercosis patients and 100% of the sera from acute cysticercosis patients. The immunodominant epitope was the carboxyl-terminal fragment of the molecule.Nucleotide sequence data reported in this paper has been submitted to the GenBank, EMBL and DDBJ databases under the accession number AJ439882     

Identification and characterization of cDNA clones encoding antigens of Eimeria tenella

An Eimeria tenella cDNA library was constructed in the expression vector λgt11 from poly (A⁺) RNA extracted from sporulating oocysts. The library was screened with rabbit antiserum raised against antigens extracted from fully sporulated oocysts. All of the antigen-expressing plaque-purified clones were initially characterized by cross screening with antisera raised against different stages of the E. tenella life cycle, as well as with antiserum raised against sporozoites of a related species, namely E. acervulina. A selected number of clones were further characterized by antibody selection coupled with immunoblotting and DNA cross hybridization. Three different E. tenella antigens were identified. All three appear to be constitutively expressed at the protein level during sporogony.     

Isolation of cDNA clones and genomic DNA clones of beta-subunit of chicken prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis. The active enzyme is a tetramer composed of two pairs of non-identical subunits, alpha and beta. Sheep antiserum directed against chicken proly 4-hydroxylase was initially used to screen two cDNA expression libraries. The cDNA was prepared from chicken smooth muscle mRNA and cloned into the plasmids pUC8- and pUC9. Antibodies identified twenty-five clones among the approximately 2 x 10(5) clones in the libraries. Ten clones were isolated pure and used in the subsequent analysis. Monospecific antibodies directed against beta subunit of the enzyme were used in Western-blot analyses of extracts of bacteria carrying the cDNA clones. The results indicated that the clone CPH 9-10B encodes a portion of beta-subunit. The cDNA from CPH 9-10B was used to screen another cDNA library prepared from mRNA from chicken skeletal muscle. Several overlapping clones were isolated. Together the cDNAs correspond to 2.4 kb which is the same as the corresponding mRNA. Three regions of the amino acid sequence deduced from the cDNA sequence matched with that of the NH2-terminus of beta-subunit and two CNBr peptides derived from beta-subunit. The cDNA of CPH 9-10B was also used to screen a genomic DNA library constructed with lambda EMBL-3. Two overlapping genomic clones lambda gCPH beta-22 and beta-50 were isolated and characterized by restriction enzyme analysis. The results indicate that lambda gCPH beta-22 contains the portion of the beta-subunit gene that is transcribed into the 5' portion of beta-subunit mRNA, whereas lambda gCPH beta-50 contains the 3' portion.     

小鼠MBL-A基因的克隆和序列分析

目的：克隆小鼠甘露聚糖结合凝集素-A(MBLA)基因的全长编码区cDNA。方法：利用RT-PCR方法，从Balb/c小鼠的肝细胞中，分离出MBL-A基因cDNA片段，克隆入pUC-T载体，测序并进行分析。结果：扩增得到的小鼠MBL-A基因cDNA全长720bp，编码240个氨基酸残基，包含了完整的富含半胱氨酸区、胶原区、颈区和糖识别域。分析表明，与Genbank中发表的序列具有99.9％的同源性。结论：获得小鼠MBL-A基因的克隆，为进一步研究MBL-A分子在体内的生物学功能奠定了一定基础。     

Human antibody recognition of Anisakidae and Trichinella spp. in Greenland

High levels of total IgE are observed among children in Greenland. To evaluate the extent to which Anisakidae and Trichinella spp. contribute to the high total IgE level, an ELISA and a western blot were developed for the detection of IgG antibodies to Anisakidae, based on excretory/secretory antigens from Anisakidae larvae. Western blots with Anisakidae and Trichinella antigens discriminated between Anisakidae and Trichinella infections, enabling cross-reactivity between the two parasite infections to be eliminated. Serum samples from 1012 children in Greenland were analysed for specific antibodies to Anisakidae and Trichinella. Eleven children were IgG-positive for Trichinella and nine were IgG-positive for Anisakidae, indicating a relatively low prevalence of both infections among children in Greenland. Faecal samples from 320 children were also examined for other intestinal parasites. Enterobius vermicularis was found in one sample and Blastocystis hominis in 32 samples, but no other intestinal parasites were identified. In total, 304 children had elevated total IgE levels. There was a significant association between Trichinella seropositivity and high levels of total IgE, but not between Anisakidae seropositivity and total IgE. The data indicate that parasitic infections alone do not explain the high level of total IgE observed among children in Greenland.     

Cloning and characterization of a cDNA specific for bovine retina

A retina-specific cDNA clone (pCR18) was selected from a bovine retinal cDNA library and characterized. The clone pCR18 consisted of 905 base pairs and hybridized to the mRNA of about 12S from the bovine retina, but not that from the brain or liver. The nucleotide sequence revealed a long open reading frame which encodes a 147 amino acid polypeptide of about 15,700 Da. No significant sequence homology with the predicted protein was found in the protein sequence library of about 3500. Messenger RNA which hybridized to pCR18 translated a polypeptide of about 19,000 Da in a reticulocyte translation system. Southern blot analysis indicated that the bovine genome contains a single copy of this gene. Furthermore, RNA dot analysis showed that the poly(A)+ RNA from the human retinoblastoma cell lines (Y79 and WERI) hybridized to pCR18, whose intensity was comparable to that of the bovine retina. In situ hybridization revealed that pCR18 was expressed mostly in some ganglion cells of the rat retina. The results suggest that cDNA clone (pCR18) encodes a protein specific for the retina and mRNA for pCR18 is mostly localized in the retinal ganglion cells and also expressed in the human retinoblastoma cells, although its function remains to be elucidated.     

Cloning, expression, and molecular characterization of the dermonecrotic toxin gene of Bordetella spp.

A cosmid library of random fragments of Bordetella bronchiseptica genomic DNA was prepared and screened with oligonucleotides designed from the sequence of the B. pertussis dermonecrotic toxin (DNT) gene. Two cosmid clones which apparently contained the complete B. bronchiseptica DNT gene were identified, but they did not express the toxin. A 5-kb fragment containing the DNT gene was subcloned from one of the cosmid clones onto a high-copy-number plasmid, and this resulted in low-level expression of the toxin. The expression level was increased by deletion of a small region upstream of the coding sequence. Assays for biological activity, including the infant mouse dermonecrosis assay, confirmed that the product of the cloned gene was DNT. The complete sequence of the B. bronchiseptica DNT gene was determined and was more than 99% homologous to the DNT gene of B. pertussis. A putative purine nucleotide-binding motif was shown to be important for toxic activity. Extracts containing the recombinant or the native toxin induced DNA synthesis in Swiss 3T3 cells but inhibited cell division leading to binucleation.     

Establishment and characterization of Epstein-Barr virus-specific human CD4+ T lymphocyte clones.

We developed a simple method for establishing Epstein-Barr virus (EBV)-specific, human CD4+ T cell clones. The method originates from our experience that the regression of cell growth in in vitro EBV transformation of B cells occurs when round lymphoid cells appear in the culture. Peripheral blood mononuclear cells (PBMCs) were cultured with EBV, and IL-2 (20 U/ml) was added to the culture on day 17 after the virus addition. The phenotype of the growing cells was CD3+, CD4+, and CD8-. The cells were cytotoxic for autologous lymphoblastoid B cell line (LCL) and EBV-superinfected autologous LCL. The cytotoxic T lymphocytes (CTLs) were confirmed to be CD4+ T cells but not CD8+ T cells in the culture. CTL clones were established by a limiting dilution method. All the CTL clones had the phenotype of CD3+, CD4+ and CD8-, and proliferated in response to autologous LCL. They produced interferon (IFN)-gamma, interleukin 2 (IL-2) and tumour necrosis factor (TNF)-beta but not IL-4. All but one clone responded to both autologous, EBV-superinfected and non-superinfected LCLs. Proliferative and cytotoxic responses to allogenic LCLs were heterogeneous. These results suggest that this method induces heterogeneous, EBV-specific CD4+ CTL clones and is useful for analysis of CD4+ T cells in EBV infections.     

Isolation and characterization of human random cDNA clones homologous to DNA from the X chromosome

To search for human X-chromosome-specific probes useful for molecular mapping, we studied recombinant clones isolated from a human cDNA library. DNA preparations from 150 randomly selected clones were labeled and annealed to XY and 4XY human DNA, and to DNA from a human-mouse hybrid cell line that had retained only the human X-chromosome (A9/HRBC2). cDNA clones sharing homology with DNA from the X chromosome annealed to A9/HRBC2-DNA and hybridized more intensely to 4XY DNA than to XY DNA. Eleven such clones were identified. Of these, three hybridized only to X chromosomal DNA while the rest also annealed to DNA from one or more autosomes. Chromosomal assignment of the autosomal DNA fragments showed that, in addition to hybridization to X chromosomal DNA, four of the clones hybridized to DNA sequences from chromosome 2 and two clones to chromosome 7. Subregional mapping of the relevant X chromosomal DNA fragments indicated that one clone is homologous to DNA sequences located at Xp21-Xp22, whereas the others are located in the telomeric region of the long arm. The cDNA clones were used to search for restriction fragment length polymorphisms. Several restriction-site polymorphisms were detected. Some corresponded to variants of X chromosomal DNA sequences while others were from autosomes such as chromosomes 2 and 7.     

Isolation of encephalitogenic CD4+ T cell clones in the rat. Cloning methodology and interferon-gamma secretion

In this report we detail a procedure for the cloning of a rat encephalitogenic T cell line and show that the methods normally employed for other species may not always be applicable. The two important differences to be described are, (i) that in these experiments where the parent T cell lines were generated with thymocytes as presenting cells, splenocytes were not suitable as a source of antigen-presenting or stimulator cells and (ii) semipurified forms of IL-2, specifically that derived from EL4 lymphoma cells, resulted in a much reduced cloning frequency and rate of T cell growth compared with cruder mixtures such as that derived from mitogen-stimulated splenocytes. Functional studies with clones derived from a strongly encephalitogenic (experimental autoimmune encephalomyelitis (EAE)-inducing) T cell line revealed that the clones had a reduced capacity to mediate EAE in recipient rats but were otherwise comparable to the parent line in terms of surface phenotype and fine antigen specificity. In an attempt to begin to identify the type of CD4+ T cells that may induce EAE we tested the clones and lines for secreted interferon-gamma by a sensitive ELISA, and showed that all clones secreted high levels of this factor.     

人TLR2和TLR4胞外区cDNA的克隆

目的 从脂多糖诱导的外周血单个核细胞克隆人Toll样受体2(TLR2)和Toll样受体4(TLR4)胞外区cDNA.方法 用不同浓度脂多糖在不同时间刺激单个核细胞后提取总RNA,RT-PCR方法半定量测定TLR2和TLR4的表达,应用pUCm-T载体克隆胞外区cDNA,双酶切以及DNA测序进行鉴定.结果 单个核细胞在四种浓度脂多糖刺激3 h,6 h和12 h后.TLR2和TLR4表达并不相同.15 μg/mL脂多糖作用6 h TLR2表达最高,10 μg/mL脂多糖作用3 hTLR4表达最高.经RT-PCR扩增TLR2和TLR4胞外区cDNA分别为1 700 bp和1 900 bp,T载体克隆、酶切鉴定及测序分析后证实,目的 片段与GenBank中序列一致.结论 脂多糖的诱导对外周血单个核细胞TLR2和TLR4表达有显著影响,并且成功克隆了胞外区cDNA.     

小鼠canstatin cDNA的克隆及其在大肠杆菌中的表达

目的:从小鼠肝脏组织克隆canstatin cDNA并在大肠杆菌(E.coli)中表达, 为进一步研究其抗肿瘤血管生成活性奠定基础。方法: 用Trizol试剂提取小鼠肝脏组织总RNA,通过RT-PCR扩增小鼠canstatin(m canstatin)的cDNA,克隆到pMD18-T载体中并进行序列分析。将小鼠canstatin cDNA 定向克隆于原核表达载体pET30a(+)中, 在大肠杆菌E.coli BL21中经IPTG诱导表达。结果:小鼠canstatin的cDNA长度为684bp,编码227个氨基酸,与已知的人canstatin的cDNA同源性为89%,氨基酸的同源性为96%。IPTG诱导原核表达载体pET30a(+)/m canstatin在大肠杆菌E.coli BL21中表达。结论: 首次成功克隆了小鼠canstatin的cDNA, 其原核表达载体pET30a(+)/m canstatin在大肠杆菌E.coli BL21中高效表达,小鼠canstatin抗肿瘤血管生成活性有待进一步研究。     

Cloning, characterization and expression analysis of pufferfish interleukin-4 cDNA: the first evidence of Th2-type cytokine in fish

Interleukin-4 (IL-4) is one of the key cytokines in Th2 mediated immune responses, which has been shown to regulate the responses of many immune cytokines, such as interferon-gamma (IFN-gamma), interleukin-1 (IL-1) and TNF-alpha. Much work on IL-4 has been done in human and several mammal species while little in fish. In this study, we have cloned and characterized the full-length cDNA of IL-4 in Tetraodon. The Tetraodon IL-4 cDNA is 834bp in length and contains a short 5'UTR of 39bp, a 3'UTR of 375bp and an open reading frame of 420bp translating into a protein of 139aa with a predicted molecular mass of 16.131kDa. The Tetraodon IL-4-encoding gene with the same organization as the mammalians and birds consists of four exons and three introns. The encoded protein shows 11-16% identities to other homologues. RT-PCR was optimized to estimate the expression level of IL-4 in Tetraodon. The results showed that IL-4 is constitutively expressed in all selected tissues, including head kidney, spleen, liver, brain, gill, muscle and heart, although low levels were observed in head kidney, spleen, and liver. The ubiquitous expression of IL-4 is consistent with a postulated role in immune cytokines regulation. Stimulating the fish with a mixed stimulant that contained 2 microg ConA, 2 microg PHA, and 2 microg PMA, significantly up-regulated the expression of IL-4 in most tissues examined, which potentially indicated that IL-4 was involved in the immune inflammatory responses triggered by mitogens. This is the first report of cloning and characterization of IL-4 cDNA and gene in fish.