首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 516 毫秒
1.
A sequence encoding bovine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been identified from a concanavalin A-stimulated bovine lymphocyte cDNA library. This sequence was isolated by hybridization with synthetic oligonucleotide probes based upon the human GM-CSF sequence. This bovine cDNA was engineered for expression and secretion of activity into the periplasmic space of E. coli. Periplasmic extracts contain a 14,500-dalton protein and stimulate colony formation of bovine bone marrow progenitor cells. The predicted protein is 70% homologous with human GM-CSF and 55% homologous with murine GM-CSF. Numerous structural features are conserved among these three proteins, such as location of cysteine residues, glycosylation sites, and overall change. The biological activity of bovine GM-CSF is species specific, since recombinant preparations do not cause proliferation of human or murine bone marrow cells. Similarly, murine GM-CSF does not exhibit activity on cells of bovine or human origin. However, human GM-CSF does stimulate colony formation of bovine bone marrow cells, although the specific activity appears reduced when compared to assays on human cells.  相似文献   

2.
CD69 is rapidly inducible on various hematopoietic cells upon stimulation and is detectable as an early activation antigen. Although CD69 is well characterized in human and mouse, no information is available on bovine CD69. We report here that, bovine CD69 was cloned from a cDNA expression library prepared from activated peripheral blood lymphocytes. The full-length cDNA contained an 80bp 5' untranslated region, followed by a 600bp coding region and AU-rich motifs in a 3' untranslated region (GenBank accession number AF272828). Comparison of the bovine CD69 coding sequence reveals 69.4 and 78.2% nucleotide sequence identities with mouse and human CD69, respectively. The predicted amino acid sequence of bovine CD69 shares 56.3 and 62.3% sequence identity when compared with mouse and human CD69, respectively. Bovine CD69 has the highly conserved amino acid sequences found in the C-type lectin family, suggesting that the conserved residues may be important for conformation and binding to the, as yet unidentified ligand. In addition, the cytoplasmic tail of bovine CD69 has two casein kinase-2 (CK-2) phosphorylation sites. These data suggest that bovine CD69 plays an important role in the activation of lymphocytes.  相似文献   

3.
In characterising Mycoplasma agalactiae strains from various European countries and from Africa, a new insertion sequence (IS), ISMag1, which is related to IS of the family of IS30 insertion elements, has been identified by DNA sequence analysis and Southern blot hybridisation. ISMag1 has a size of 1515bp, and contains inverted repeats of 3bp and a gene encoding the putative transposase on a single open reading frame. ISMag1 is present only in the rarely isolated serotypes E, F, G and H of M. agalactiae, where it is found in 1 to approximately 30 copies. The different patterns obtained by hybridisation of a labelled probe of ISMag1 to genomic DNA cut with various restriction enzymes correlate to some extent to the different serotypes and to variations of the nucleotide sequences of the uvrC genes of the different strains. Based on uvrC sequences, the strains of M. agalactiae carrying ISMag1 form a cluster, separate from the other strains. IS patterns obtained with ISMag1 allow a fine subtyping of the serotypes E, F, G and H of M. agalactiae for epidemiological studies. The potential role of ISMag1 and of its copy numbers on virulence and persistence of the respective strains requests further studies.  相似文献   

4.
Ⅶ型分泌系统(T7SS)是近年来发现的分泌系统,分泌两种胞外蛋白,EsxA基因编码的ESAT6蛋白和EsxB基因编码的CFP-10蛋白。分泌蛋白具有良好的免疫原性,促进细菌从巨噬细胞吞噬体逃逸、影响巨噬细胞凋亡及裂解细胞等生物学功能,与致病性密切相关。以罗非鱼源无乳链球菌为模板,克隆到294bp的EsxA基因,将目的序列克隆到pMD18-T载体,经测序,目的序列与无乳链球菌EsxA基因同源率在99%以上。EsxA基因克隆到pET32a载体中,重组载体在28℃、0.5mmol/L IPTG诱导条件下表达量最大,可溶性表达。将纯化的ESAT6蛋白免疫Balb/c鼠,成功制备ESAT6蛋白鼠多克隆抗体,经Western blot,ESAT6蛋白具有良好的反应原性。研究结果为进一步进行无乳链球菌ESAT6蛋白的免疫学功能研究奠定了基础。  相似文献   

5.
Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with HRMA can be used as a powerful, fast and low-cost tool for the differentiation of clinically important bacterial mastitis pathogens.  相似文献   

6.
GTPases of the Rho family play important roles in human leukocyte signal transduction pathways; however, little is known about the function of these proteins in bovine cells. In the present studies, we isolated molecular clones of bovine Rac1, Rac2, and the Rac/Rho GTPase regulatory protein D4-GDP dissociation inhibitor (D4-GDI) from a bovine bone marrow cDNA library. These clones contained complete open reading frames, encoding 192, 192, and 200 amino acids, respectively. Comparison of the bovine amino acid sequences with those of other species demonstrated a high degree of identity of these proteins across all species, suggesting that these proteins likely play conserved functional roles in bovine leukocyte signal transduction pathways. Comparative Western blotting of these proteins in human and bovine neutrophil cytosol demonstrated that Rac2 was the predominant Rac species and that D4-GDI was the predominant GDI species in bovine neutrophil cytosol. Despite the high degree of homology between human and bovine Rac2, some of the anti-peptide antibody probes prepared against human Rac2 failed to recognize the bovine homologue. We also showed by subcellular fractionation techniques that Rac2 is localized primarily to the cytosolic compartment of resting bovine neutrophils, but is translocated to the plasma membrane after stimulation with PMA. These findings suggest that Rac2 does play a role in bovine neutrophil activation. In addition, these data will be helpful in developing more specific probes for investigating the role of these proteins in bovine leukocyte signal transduction pathways and for studying various inflammatory diseases in cattle.  相似文献   

7.
CD97 is a member of a novel subfamily of leukocyte proteins that are characterized by the presence of tandemly repeated extracellular epidermal growth factor (EGF)-like domains and a seven-span transmembrane region, known as EGF-TM7. We here report the cloning of cDNA encoding the pig homologue of CD97. A pig CD97 specific probe was generated by PCR amplification of pig leukocyte cDNA, using primers based on consensus regions among the known sequences of mouse and human CD97. Screening of a pig aorta smooth muscle cDNA library identified one clone containing an open reading frame (ORF) that encoded an 18 amino acid putative signal peptide, a 141 amino acid sequence consisting of three EGF domains, a mucin-like spacer region of 276 amino acid, containing a G-protein coupling motif of 52 amino acids, followed by a 250 amino acid region containing seven membrane spanning domains and a 47 amino acid cytoplasmic tail. The amino acid sequence of the clone was 75, 67 and 59% homologous to cattle, human and mouse CD97 antigen, respectively. Therefore, it was termed pig CD97. Pig CD97 antigen shares many structural features with human, cattle and mouse CD97. RT-PCR analysis of cDNA from different pig cells and tissues showed that CD97 was highly expressed in leukocytes and lymph node cells. This is the first report describing the identification of a member of the EGF-TM7 family in the pig.  相似文献   

8.
  相似文献   

9.
为了探索牛乳腺炎无乳链球菌磷酸甘油酸激酶（Phosphoglycerate kinase,Pgk）基因编码蛋白的抗原性,根据GenBank公布的牛源无乳链球菌Pgk基因序列,设计并合成一对引物,通过PCR扩增出Pgk蛋白抗原优势区的编码序列,并对其进行克隆、测序、转化、诱导表达及抗原性鉴定。结果显示克隆的Pgk基因序列含有984 bp,编码328个氨基酸残基。临床分离株与GenBank上公布的B群无乳链球菌菌株Pgk基因（AE009948）核苷酸序列同源性为99.09%,氨基酸序列同源性为99.69%。将扩增的Pgk基因片段克隆至原核表达载体pET30a（＋）中,构建重组表达载体Pgk-pET30a（＋）,再将筛选的阳性重组质粒转化至BL21工程菌,经IPTG诱导获得部分可溶性表达Pgk重组蛋白。经Ni2＋亲和层析柱纯化得到纯度在90%以上的蛋白,经Western blot分析结果表明,重组蛋白具有良好的抗原性,为进一步探索其对奶牛的免疫原性研究奠定了良好的实验基础。  相似文献   

10.
An enzyme-linked immunosorbent assay (ELISA) demonstrated the presence of naturally acquired antibodies against Streptococcus agalactiae in normal bovine serum (NBS). In milk wheys, ELISA values were much lower than in sera. Pre-colostral calf serum (PCS) was shown to lack antibodies to type II and III S. agalactiae. The opsonic requirements of 10 human and 10 bovine strains were investigated by evaluating the phagocytosis-induced reduction of the incorporation of radiolabeled thymidine by streptococci. Antibodies present in NBS were required for the efficient ingestion of both human and bovine isolates type II by bovine granulocytes. Three out of five type III bovine isolates were opsonized in the absence of specific antibodies (opsonization by PCS) and type II and III bovine isolates did not require complement opsonization. By contrast, inactivation of complement reduced phagocytosis of human isolates and only one type III strain of human origin was opsonized by PCS. These findings suggest that human isolates had higher opsonic requirements. The phagocytic killing of 6 type III strains (5 mastitis isolates and the reference typing strain) was investigated. Opsonization by normal serum enabled bovine blood granulocytes to ingest and kill S. agalactiae. Nevertheless, greater than or equal to 35% of bacteria remained viable at the end of the phagocytosis incubation in 10% NBS. Heat treatment of serum decreased the efficacy of killing for only 3 of the 6 tested strains. An IgG2 fraction of normal adult bovine serum promoted active ingestion, which was still increased in the presence of PCS. Normal wheys displayed large variations in their ability to promote ingestion of S. agalactiae by blood granulocytes. The promoting effect was systematically less than that of serum from the same cow, and this can be related to the lower ELISA values found in wheys.  相似文献   

11.
Human P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric membrane mucin expressed on leukocytes that binds selectins. Here, we report that the open reading frame (ORF) of bovine PSGL-1 (bPSGL-1) cDNA is 1284 base pairs in length, predicting a protein of 427 amino acids including an 18-amino-acid signal peptide, an extracellular region with a mucin-like domain, and transmembrane and cytoplasmic domains. The amino acid sequence of bPSGL-1 demonstrated 52, 49 and 40% overall homology to equine, human and mouse, respectively. A single extracellular cysteine, at the transmembrane and extracellular domain junction, suggests a disulfide-bonding pattern. Alignment of bovine with equine, human and mouse PSGL-1 demonstrates high conservation of transmembrane and cytoplasmic domains, but diversity of the extracellular domain, especially in the anionic NH(2)-terminal of PSGL-1, the putative P-selectin binding domain. In the NH(2)-terminal of bPSGL-1, there are three potential tyrosine sulfation sites and three potential threonine O-glycosylation sites, all of which are required for P-selectin binding in human PSGL-1 (hPSGL-1). bPSGL-1 shares only 57% homology in amino acid sequence with the corresponding epitope region which binds the monoclonal antibody PL1 for hPSGL-1, and no cross-reactivity was found in bovine leukocytes. In summary, bPSGL-1 shares homology with hPSGl-1, but has differences in the putative extracellular P-selectin binding domain.  相似文献   

12.
IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full‐length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig‐like domain showed that Ig‐like domain effectively prevented pig sperm–egg interactions.  相似文献   

13.
Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis were examined for their ability to interact with 125I-labelled fibronectin, fibrinogen and type II collagen. Their relative surface hydrophobicity and production of extracellular capsule were also investigated. Almost all S. aureus strains bound fibronectin (mean value 23%), fibrinogen (mean value 12%) and type II collagen (mean value 16%). CNS bound fibronectin (mean value 6%) and type II collagen (mean value 7%), but not fibrinogen (mean value 2%). The specificity of binding of these proteins to S. aureus strain F1440 and to coagulase-negative Staphylococcus chromogenes strain BO52 was studied by adding an excess of unlabelled proteins. Fibronectin and collagen binding were observed to be specific, varying between 50 and 75%, whereas the specificity of fibrinogen binding to S. aureus strain F1440 was lower (26%). Most of the S. aureus strains (63%) showed very high surface hydrophobicity (autoaggregation) or lower hydrophobicity (29% of the strains) and the rest were hydrophilic. None of the CNS strains autoaggregated, 44% were classified as hydrophilic strains. Hydrophilic strains (except the reference strains) did not show extracellular capsule production. However, the encapsulated (reference) strains showed low binding to these proteins as compared to their unencapsulated variants. Pre-treatment of S. aureus strain F1440 and S. chromogenes strain BO52 with trypsin decreased their fibronectin binding capacity and surface hydrophobicity, whereas pre-treatment with bovine milk (except on collagen binding to strain F1440) did not significantly affect binding to these proteins. These data indicate that S. aureus and CNS isolated from bovine udder infection have the ability to bind to tissue matrix and plasma proteins which may be exposed in the traumatized or toxin-damaged udder epithelial lesions.  相似文献   

14.
Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of attaching and effacing (AE) lesions on intestinal epithelial cells. Secretion of extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the AE lesions by human EPEC. In this study, we show that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system. Bovine EPEC and EHEC strains present two different secretion profiles of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR. We also demonstrate that genes encoding secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69.  相似文献   

15.
The detection of bovine lactoferrin binding protein on Trypanosoma brucei   总被引：1，自引：0，他引：1  
Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth. Therefore, T. brucei has a TF receptor that allows it to obtain iron from TF. Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals. LF has been shown to interact with some bacteria species by specific receptor-ligand binding. We examined the ability of T. brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system. We found that bLF bound to components of T. brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins. Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T. brucei, which exhibited molecular masses of 40 and 43 kDa. The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).  相似文献   

16.
  相似文献   

17.
The full length of gene encoding Fc receptor types III for bovine immunoglobulin G (boFcgammaRIII, CD16) was obtained with PCR from the c DNA library of bovine alveolar macrophages. Through molecular cloning and sequencing it is shown that the coding gene has 507 bp and codes for 168 amino acids. There are signal peptide, transmembrane spanning domain and three potential N-linked glycosylation sites in the sequence. Compared with the boFcgammaRIII cDNA sequence cloned by Collins et al, it deletes 82 amino acids and has only one extracellular domain. It is suggested that this cDNA fragment is a special form of the gene structure of bovine FcgammaRIIIA (CD16-II).  相似文献   

18.
  相似文献   

19.
Bovine interleukin 2: regulatory mechanisms   总被引：1，自引：0，他引：1  
A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.  相似文献   

20.
The cDNA clone of bovine pim-1 has been isolated from phorbol-12-myristate-13-acetate (PMA) and concanavalin A (ConA)-activated peripheral blood lymphocytes (PBLs). The full-length cDNA contains a 411bp 5' untranslated region (5'-UTR), followed by a 939bp coding region and a 3' untranslated region (3'-UTR) that contains 1403bp. Comparison of the bovine pim-1 coding sequence with the human, rat, mouse, frog and zebrafish counterparts reveals 94, 90, 89, 67 and 40% homology at the nucleotide level, respectively. The predicted amino acid sequence of bovine Pim-1 shares 98.7, 97.1, 93.3, 68.8, and 52.4% similarity with the sequences of human, rat, mouse, frog, and zebrafish, respectively. The 5'-UTR of bovine pim-1 shares high sequence similarity to the human and mouse counterparts and is G/C-rich (75%) which may promote a high degree of secondary structure. The 3'-UTR of bovine pim-1 contains two potential polyadenylation sites and an A/T-rich motif which has been shown to decrease the stability of polyA mRNA molecules. Southern blot results indicate that a single copy of the gene exists in the bovine genome. Northern blot results show that PMA stimulation of PBLs increases the expression of the pim-1 mRNA. In addition, examination of Pim-1 protein expression in PBLs stimulated with a variety of mitogens including ConA, PMA, anti-CD3 and purified protein derivative (PPD) from Mycobacterium tuberculosis, reveals two different types of expression patterns during the course of a 24h period of stimulation. ConA and PPD gave a biphasic pattern of expression while PMA and anti-CD3 gave single transient pattern of expression suggesting that expression is controlled by more than one signaling pathway.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号