共查询到18条相似文献，搜索用时 250 毫秒
1.
为分析禽呼肠病毒(ARV)标准毒株S1133株感染鸡胚成纤维细胞(CEF)后对相关鸡Toll样受体(ChTLRs)mRNA转录水平的影响作用,利用实时荧光定量PCR,测定和分析ARV-S1133感染CEF后ARV结构蛋白σC和ChTLRs的mRNA转录水平变化情况。结果表明,ARV-S1133感染CEF 10h后,ARVσC蛋白的mRNA相对表达量开始迅速上升,在48h达到峰值;同时,感染CEF中的ChTLR3、ChTLR5、ChTLR7、ChTLR15和ChTLR21mRNA表达量发生显著变化,在感染72h内各个受体的mRNA表达量呈波浪式变化。5个不同滴度的ARV感染CEF 24 h后,ChTLR3、ChTLR5、ChTLR7、ChTLR15、ChTLR21mRNA转录水平与病毒滴度均呈正线性相关。上述结果表明,ARV感染后可诱导CEF ChTLR3、ChTLR5、ChTLR7、ChTLR15、ChTLR21的mRNA转录水平发生变化,可能与禽呼肠病毒的复制和致病机制相关。 相似文献
2.
雏鸡不同组织TLR1、TLR2、TLR4、TLR5和TLR15 mRNA转录水平相对定量研究 总被引：1，自引：0，他引：1
参考GenBank登录的ChTLRs基因序列设计实时定量PCR特异性引物,建立检测鸡Toll样受体(ChTLR)mRNA相对转录水平的实时定量PCR方法,分析ChTLR1、ChTLR2、ChTLR4、ChTLR5和ChTLR15在雏鸡不同器官组织中的转录水平。结果显示5种ChTLRs在脾脏、法氏囊、胸腺和各段肠道组织中均有转录。其中,ChTLR1 mRNA在法氏囊、肾脏和盲肠组织中转录水平较高;ChTLR2 mRNA在脾脏、法氏囊和肝脏等组织中转录水平较高,在肾脏、肺脏和皮肤未检测到转录;ChTLR4 mRNA在所检测组织中转录水平差异较小,在脾脏、十二指肠和胸腺转录水平较高;ChTLR5 mRNA在肾脏、脾脏和空肠中的转录水平较高;ChTLR15 mRNA在法氏囊中转录水平最高,其次为脾脏和盲肠。本研究建立了检测ChTLRs mRNA在不同器官组织中表达水平的实时定量PCR方法,ChTLRs mRNA在雏鸡各器官组织中转录水平差异较大,可能与雏鸡各器官组织对病原的识别和抵抗能力有关。 相似文献
3.
为研究病毒与机体的相互作用,本研究参考GenBank中鸡Toll样受体21 (ChTLR21)的基因序列设计实时定量PCR特异性引物,以鸡核糖体蛋白L4 (RPL4)为内参基因,建立检测ChTLR21 mRNA相对转录水平的实时定量PCR方法,分析ChTLR21在禽传染性喉气管炎病毒(ILTV)感染的鸡胚成纤维细胞(CEF)中和感染SPF雏鸡免疫器官脾脏、法氏囊和胸腺组织中的转录水平.结果显示:ILTV感染CEF后2h、4h、8h和18h时间点ChTLR21 mRNA转录水平分别为未感染对照细胞的1 540.53、0.98、1.19和3.70倍.但仅在ILTV感染2h时引起ChTLR21转录水平升高,与对照组相比差异显著(P＜0.05).ILTV感染SPF雏鸡后6h、24 h和30 h脾脏中ChTLR21 mRNA转录水平分别为未感染对照的56.34、59.85和3.61倍;法氏囊中分别为0.03、25.98和3.08倍;胸腺中分别为2.52、50和7.32倍.在感染初期,脾脏中ChTLR21转录量显著升高,随后有所降低,但均高于未感染对照(p＜0.05);法氏囊中仅在感染6h时呈显著抑制(p＜0.01);胸腺中呈波动性转录水平升高,但与对照组无统计学差异(p＞0.05).本研究证明ChTLR21参与了鸡体对ILTV感染的应答,并在体内外感染模型中呈现不同的表达规律. 相似文献
4.
雏鸡不同组织TLR3、TLR7和TLR21 mRNA转录水平研究 总被引：1，自引：0，他引：1
参考GenBank登录的ChTLRs基因序列设计实时定量PCR特异性引物,建立检测鸡T011样受体（ChTLR）mRNA相对转录水平的实时定量PCR方法,分析ChTLR3、ChTLR7和ChTLR21在雏鸡不同器官组织中的转录水平。3种ChTLRs在脾脏、法氏囊、胸腺和各段肠道组织中均有转录。其中,ChTLR3 mRNA在肾脏、胸腺、盲肠、空肠、肝脏和十二指肠转录水平较高,在皮肤中未检测到转录;chTLR7 mRNA在脾脏、肾脏、盲肠、胸腺和十二指肠转录水平较高,在肝脏和皮肤中转录水平很低;ChTLR21 mRNA在所检测组织中均有转录,其中在脾脏转录水平最高,其次为法氏囊、胸腺和十二指肠。结果表明,ChTLRs mRNA在雏鸡各器官组织中转录水平差异较大,可能与雏鸡各器官组织对病原的识别和抵抗能力有关。 相似文献
5.
为探究鸡TLR1A基因正选择nsSNP位点与沙门氏菌易感性的关联，挖掘其潜在的功能性位点，本研究利用多种生物信息学方法对ChTLR1A的21个nsSNP位点及基因的选择压力进行综合分析，筛选ChTLR1A正选择nsSNP功能性位点，深入分析其与鸡沙门氏菌易感性之间的相关性。结果显示，rs739087452(I388L)和rs313678806(C815R)在种上及种内均受到正选择作用，且rs739087452(I388L)位点的变异使其蛋白稳定性降低。rs313678806(C815R)与鸡沙门氏菌的易感性显著相关（P<0.001）,C/C基因型为沙门氏菌抵抗型，表明rs313678806(C815R)可能是影响鸡沙门氏菌易感性的重要功能性SNP。本实验结果可为TLR1A基因标记在鸡抗病育种中的利用提供参考依据。 相似文献
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7.
通过ELISA和IHC分别检测了乳腺肿瘤患犬血清和组织中CA15-3的浓度和表达情况,发现CA15-3在乳腺肿瘤患犬血清浓度(15. 70 U/m L±12. 93 U/m L)明显高于健康犬血液中CA15-3浓度(5. 72 U/m L±4. 82 U/m L)。IHC结果发现,CA15-3 MOD在乳腺肿瘤组织中(0. 21±0. 08)明显高于健康乳腺组织(0. 10±0. 04),且在不同的组织分级中差异显著(P 0. 05)。结论:血清中CA15-3浓度可作为犬乳腺肿瘤早期诊断和预后评估的生物标志物之一,组织中CA15-3蛋白可作为评估犬乳腺肿瘤恶性程度的生物标志物之一。 相似文献
8.
为研制犬副流感特异性诊断试剂,我们以犬副流感病毒(CPIV)免疫8周龄BALB/c小鼠,采用淋巴细胞杂交瘤技术获得4株稳定分泌针对CPIV的单克隆抗体(MAb)细胞株,分别命名为4F386、584C9、4G7F4和4C9D8.4株MAb腹水针对CPIV的间接ELISA抗体效价达1:10~5～1:10~6,与犬瘟热病毒(CDV)和犬细小病毒(CPV)均不发生交叉反应.MAb 4F386和4C9D8为IgG,5B4C9和4G7F4为IgM.Western blot检测表明,4F386与CPIV的F蛋白发生特异性反应,4G7F4与CPW的HN蛋白发生特异性反应,而584C9和4C9D8不与变性的CPIV蛋白发生反应.4株MAb均具有中和病毒活性,间接免疫荧光检测均呈为阳性.本研究为进一步研制CPIV特异性诊断和治疗制剂创造了条件. 相似文献
9.
我市某场外购一批仔猪162头,饲养1周后发生猪瘟与猪肺疫混合感染,采用中、西药结合防治,获得良好的疗效. (一)发病情况邵武市城郊镇某养猪场于2007年3月12日从浙江衢州购进一批杜长太三元杂交仔猪162头,平均体重12kg. 相似文献
10.
采用重氮化法合成磺胺二甲嘧啶(SM_2)-人血清白蛋白(HSA)免疫抗原和SM_2~-卵清白蛋白(OVA)包被抗原。经紫外光谱扫描法确认SM_2与载体蛋白偶联成功；经计算SM_2与HSA、OVA的结合比分别为9：1和15：1。利用杂交瘤技术和有限稀释法经过5次亚克隆,得到三株特异性稳定分泌SM_2抗体的杂交瘤细胞,经鉴定该单克隆抗体免疫球蛋白亚类为IgG_1,为入链,分子量为162Ku,染色体数目90条左右,亲和常数为6．1×10~(12)M~(-1)。与其他四种磺胺药和两种载体蛋白HSA、OVA均无交叉反应。 相似文献
11.
Porcine reproductive and respiratory syndrome virus (PRRSV) was divided into North American and European genotypes. NSP1 was an important non-structural protein of PRRSV, which was auto-cleaved from the replicase polyprotein into NSP1α and NSP1β subunits and played an important role in the immune suppression. In this study, six monoclonal antibodies (MAbs) against the recombinant PRRSV NSP1, expressed in Escherichia coli system, were screened out and identified. Western blot and IFA results indicated that 4 out of 6 MAbs recognized the recombinant NSP1α and 2 MAbs recognized NSP1β. Epitope mapping results indicated that MAb 4H2 recognized the linear epitopes E(54)EPLRW(59) in NSP1α, MAbs (2G5, 3E11 and 4D4) recognized the epitopes H(157)VLTNLP(163) in NSP1α, and MAbs 3C7 and 1H7 reacted with the epitopes 185aa to 232aa in NSP1β. Protein sequence alignment of NSP1 indicated E(54)EPLRW(59) was conserved in all North American PRRSV strains, whereas European type strains has variable amino acids in this region. The epitope H(157)VLTNLP(163) was relatively conserved among all PRRSV strains, except for a L162→S162 change in European type strains. The epitope 185-232aa was variable among North American PRRSV strains. These results may facilitate future investigations into the function of NSP1 of PRRSV and diagnostic methods for PRRSV infection. 相似文献
12.
为了探究我国地方鸡种TLR4基因的多态性和群体遗传特性,本研究采用PCR-SSCP及DNA测序方法,对我国12个地方品种鸡TLR4基因部分序列的单核苷酸多态性进行分析.结果显示,在所有鸡种TLR4基因的第二内含子上均发现了G1894C的突变.除了固始鸡极显著偏离Hardy-Weinberg平衡(P＜0.01)外,其他品种均处于Hardy-Weinberg平衡(P＞0.05).12个鸡品种该位点的多态信息含量均属于高度多态,其中皖南三黄鸡该位点的多态信息含量和杂合度最高,北京油鸡次之,文昌鸡最低;12个品种间的基因型分布存在极显著差异(P＜0.01).本研究表明,中国地方鸡种TLR4基因G1894C位点多态丰富,为深入开展鸡TLR4基因抗病育种的研究提供了一定的理论基础. 相似文献
13.
Production and characterization of monoclonal antibodies against porcine interleukin-4 总被引：1，自引：0，他引：1
Ishikura Y Kato H Hashimoto T Nishimura Y Iwata H 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2005,67(5):503-508
Interleukin 4 (IL-4) is an important regulatory cytokine produced by activated T lymphocytes and mast cells, and regulates the growth and differentiation of cells such as B and T lymphocytes. In the present study, recombinant thioredoxin (Trx)-porcine IL-4 (pIL-4) fusion protein was prepared by Escherichia coli (E. coli), and by using this protein as an immunogen, monoclonal antibodies (mAbs) against pIL-4 were produced to establish a basis for a research on immune responses in pigs. Six stable hybridoma cell lines were successfully established and specific binding of each mAb to recombinant pIL-4 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that the subclass of 5 out of 6 mAbs was IgG1 and the rest was IgG2b. Further, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to 4 different epitopes. The recombinant proteins and mAbs produced in this study will be useful tools for the assessment of porcine immune system. 相似文献
14.
Matsubayashi M Kimata I Iseki M Lillehoj HS Matsuda H Nakanishi T Tani H Sasai K Baba E 《Veterinary parasitology》2005,128(1-2):47-57
In a previous study, we have developed several chicken monoclonal antibodies (mAbs) against Eimeria acervulina (EA) in order to identify potential ligand molecules of Eimeria. One of these mAbs, 6D-12-G10, was found to recognize a conoid antigen of EA sporozoites and significantly inhibited the sporozoite invasions of host T lymphocytes in vitro. Furthermore, some of these chicken mAbs showed cross-reactivities with several different avian Eimeria spp. and the mAb 6D-12-G10 also demonstrated cross-reactivities with the tachyzoites of Neospora caninum and Toxoplasma gondii. Cryptosporidium spp. are coccidian parasites closely related to Eimeria spp., and especially C. parvum is an important cause of diarrhea in human and mammals. In the present study, to assess that the epitopes recognized by these chicken mAbs could exist on Cryptosporidium parasites, we examined the cross-reactivity of these mAbs with Cryptosporidium spp. using an indirect immunofluorescent assay (IFA) and Western blotting analyses. In IFA by chicken mAbs, the mAb 6D-12-G10 only showed a immunofluorescence staining at the apical end of sporozoites of C. parvum and C. muris, and merozoites of C. parvum. Western blotting analyses revealed that the mAb 6D-12-G10 reacted with the 48-kDa molecular weight band of C. parvum and C. muris oocyst antigens, 5D-11 reacted the 155 kDa of C. muris. Furthermore, these epitopes appeared to be periodate insensitive. These results indicate that the target antigen recognized by these chicken mAbs might have a shared epitope, which is present on the apical complex of apicomplexan parasites. 相似文献
15.
本研究旨在探明鸡恒定链（invariant chain，Ii）与内吞体转运蛋白Rab5a和Rab7b结合的结构域和在细胞内共定位的特征。首先，用PCR和基因突变技术将Ii胞浆区与跨膜区[Ii（Cyt-Tra）]、Ii CLIP （class Ⅱ-associated invariant chain peptide）-三聚体区[Ii（CLIP-TRIM）]和Ii突变体[Ii（M81-87aa）、Ii（M91-99aa）和Ii（M81-99aa）]分别插入pET-32a和pEGFP-C1构建相应的原核和真核重组质粒。其次，将构建的含有绿色荧光蛋白的重组质粒与实验室保存的含有红色荧光Rab5a和Rab7b的重组质粒共转染至人胚胎肾细胞系293 T，观察它们的共定位。将构建的原核重组质粒进行表达和纯化，最后用拉下法和免疫印迹检测Ii与Rab5a和Rab7b的结合域。结果表明，成功构建Ii结构域及Ii突变体的重组质粒。Ii（Cyt-Tra）及Ii突变体均能与Rab5a和Rab7b在细胞内共定位，而Ii（CLIP-TRIM）与空载体却不能。Ii的胞浆区和跨膜区是与Rab5a和Rab7b结合的功能结构域，而不是CLIP与三聚体区。综上所述，鸡Ii与Rab5a和Rab7b共定位和结合的区域是其胞浆区和跨膜区，而不是内质网腔区。这些结果提示Rab分子参与了Ii在胞内细胞器的转运机制，为进一步研究Ii及其载体在细胞内的转运机制和功能提供了新的途径。 相似文献
16.
We recently cloned the genes encoding chicken IL-15 and IL-15 receptor (R) alpha proteins. In this study, 12 monoclonal antibodies (mAbs) against recombinant chicken IL-15Ralpha were produced and characterized. By enzyme-linked immunosorbent assay (ELISA), all mAbs showed binding specificity for IL-15Ralpha, but not IL-2 or interferon-gamma, and identified a 25.0kDa protein by immunoblot analysis. Flow cytometric analysis revealed negligible expression of IL-15Ralpha on non-activated lymphocytes from the spleen, thymus or bursa, low but detectable expression on macrophages and high expression on concanavalin A-activated spleen lymphoblasts. Established chicken T cell (RP13) and macrophage (HD11) cell lines expressed substantially higher levels of IL-15Ralpha compared with a B cell line (RP9). Two mAbs inhibited IL-15 dependent proliferation of T cells suggesting that the tertiary structure of the protein domain of native IL-15Ralpha that binds to IL-15 is preserved in the recombinant receptor molecule. These mAbs will be useful reagents for further in vitro and in vivo studies of the biological functions of chicken IL-15 and its receptor. 相似文献
17.
Monoclonal antibodies (mAbs) against a recombinant carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis were produced in mice. Two of the mAbs cross-reacted with Mycobacterium avium and Mycobacterium intracellulare in both an elisa and immunoblot. The recombinant protein also reacted with polyclonal sera produced in rabbits against all three mycobacteria, indicating the presence of cross-reactive epitopes in the protein. To determine the reactivity of cattle sera against epitopes recognised by the mAbs, competition assays between bovine sera and the mAbs were carried out. Two mAbs were significantly inhibited by sera from cattle that were naturally infected with M paratuberculosis. The results indicate that epitopes on the carboxyl terminus of the 34 kDa protein common to M paratuberculosis, M avium and M intracellulare readily induce antibody production in naturally infected cattle. These epitopes reduce the diagnostic specificity of the carboxyl terminus of the 34 kDa protein, which was originally thought to contain only M paratuberculosis-specific epitopes. 相似文献
18.
Production of antibodies against chicken interferon-gamma: demonstration of neutralizing activity and development of a quantitative ELISA 总被引：4，自引：0，他引：4
Lambrecht B Gonze M Meulemans G van den Berg TP 《Veterinary immunology and immunopathology》2000,74(1-2):137-144
Four monoclonal antibodies (mAbs) specific for chicken interferon-gamma (ChIFN-gamma) were generated by gene gun immunization and were utilized to develop a mAb-based capture ELISA specific for ChIFN-gamma. Each mAb reacted specifically with both baculovirus and Escherichia coli-derived recombinant ChIFN-gamma in ELISA and Western Blot analysis or natural ChIFN-gamma in immunofluorescence experiments. As determined by competition ELISAs, mAbs 3D5, 4C6 and 3A3 recognized the same or adjacent epitopes on the ChIFN-gamma molecule, whereas mAb 1E12 recognized a distant epitope. Moreover, this latter mAb was able to highly neutralize the biological activities of both recombinant and natural ChIFN-gamma as measured by inhibition of viral replication and macrophage activation. To improve the detection of ChIFN-gamma, a capture ELISA was developed using mAb 1E12 as capture antibody and biotinylated mAb 4C6 as detection antibody. In addition to being more rapid and easier to perform than classical cell-mediated immunity tests, this ELISA has excellent sensitivity and improved specificity. The use of a specific rabbit polyclonal serum as revealing antibody further increased the sensitivity of the detection down to 0.5ng/ml of ChIFN-gamma. This ELISA would provide a sensitive tool to measure the in vitro release of ChIFN-gamma by T-cells in response to specific recall antigen. 相似文献