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1.
Presence of the di-leucine motif in the cytoplasmic tail of the pig FcRn alpha chain 总被引：2，自引：0，他引：2
Zhao Y Kacskovics I Zhao Z Hammarström L 《Veterinary immunology and immunopathology》2003,96(3-4):229-233
The sequence of the pig FcRn alpha chain was recently published. The lack of a conserved di-leucine motif in the cytoplasmic tail suggests a rare polymorphism in the described animal, alternatively, a sequencing error. We therefore cloned and sequenced the pig FcRn alpha chain. Our sequence, along with a previous NCBI GenBank submission and five pig derived EST clones clearly demonstrate the presence of di-leucine motif in the cytoplasmic tail of the pig FcRn. No polymorphism in the cytoplasmic tail-encoding region was found in 25 animals from six pig breeds based on single-stranded conformation polymorphism and sequencing analysis, suggesting that the previously described pig FcRn alpha chain may represent a sequencing error in the 3' portion of the gene. 相似文献
2.
Localization of the sheep FcRn in the mammary gland 总被引：5，自引：0，他引：5
Mayer B Zolnai A Frenyó LV Jancsik V Szentirmay Z Hammarström L Kacskovics I 《Veterinary immunology and immunopathology》2002,87(3-4):327-330
Among the multiple functions, which have been identified for the neonatal Fc receptor (FcRn), we study its role in the IgG transport in the mammary gland during the colostrum formation. For this reason, we have obtained several mammary gland biopsies from a pregnant sheep around parturition. The presence of the FcRn heavy chain mRNA was detected exclusively in the acinar and ductal epithelial cell by in situ hybridization (ISH). We detected strong signal in samples harvested 24 and 10 days prepartum; however, in samples we collected postpartum was barely detectable. Immunohistochemistry confirmed our ISH data. The cytoplasm of the epithelial cells of the acini and ducts in the mammary gland biopsies stained homogeneously before parturition, although a remarkable difference was observed in the pattern after lambing. The signal indicated uneven distribution of the FcRn alpha chain in the epithelial cells 1 and 5 days postpartum, since the apical sides of the epithelial cells were highlighted. The presence of the FcRn in the acinar and ductal epithelial cells and the obvious change of its distribution before and after parturition suggest that FcRn plays an important role in the IgG transport during colostrum formation. FcRn expression was also found in the lamb duodenal crypt epithelial cells, which have been previously demonstrated to secrete IgG1 in newborn ruminants, suggesting secretory role of the FcRn in ruminant epithelial cells. 相似文献
3.
本试验对贵州关岭牛解偶联蛋白3（uncoupling protein,UCP3）基因5'侧翼区进行克隆,并利用生物信息学软件对其1080 bp的克隆序列进行分析,以研究UCP3基因5'侧翼区特异性转录调控元件的调控机制。软件分析发现关岭牛UCP3基因5'侧翼区含有6个可能的转录起始点和33个潜在转录因子结合位点,与东北虎、家猫、印度水牛、藏羚羊、山羊、绵羊UCP3基因5'侧翼区（-196～+100 bp）序列相似性分别为82%、82%、98%、95%、96%和98%;该区域保守性较强,推测转录起始点上游-200～+1 bp可能是其核心启动子区域,该区域在调控基因转录和翻译方面具有共同的作用。试验成功克隆了UCP3基因5'侧翼区序列1080 bp,初步预测了该基因的核心启动子区。 相似文献
4.
Chen L Li M Li Q Yang X An X Chen Y 《Veterinary immunology and immunopathology》2008,124(3-4):284-294
To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals. 相似文献
6.
【目的】确定银黑狐黑素皮质激素受体1（melanocortin 1 receptor，MC1R）基因的核心启动子区，为探究该基因的表达调控机制提供理论依据。【方法】以银黑狐基因组DNA为模板，PCR扩增获得MC1R基因5'-UTR区序列，利用3个在线生物学软件综合预测MC1R基因启动子活性区；PCR扩增获得该基因5'-UTR区不同长度的缺失片段，并克隆至pMD19-T载体，将重组质粒转染至黑色素B16细胞中，利用双荧光素酶检测技术对10个缺失片段进行荧光素酶活性检测。【结果】成功获得银黑狐MC1R基因5'-UTR区序列，软件预测显示－596/＋73 bp可能为启动子活性区。双荧光素酶活性检测发现，构建的10个缺失片段的荧光素酶表达载体均具有启动子活性，其中pGL3-MC1RP8（－520/＋73 bp）有较强的活性，提示其为核心启动子区，与软件预测结果基本一致。【结论】试验确定了银黑狐MC1R基因的核心启动子区为―520/＋73 bp，为深入研究该基因的毛色调控机制奠定了理论基础。 相似文献
7.
Sequence and expression of the FcRn in the porcine mammary gland 总被引：7，自引：0，他引：7
9.
【目的】 鉴定绵羊趋化因子C-C基序配体19(C-C motif chemokine ligand 19,CCL19)基因启动子的核心启动子区域和关键转录因子,探究该基因在转录调控方面的作用机制。【方法】 选取绵羊CCL19基因5'-侧翼序列1 000 bp,PCR扩增启动子的7个不同长度的截短片段,并连接至pGL3-Basic质粒;将重组质粒与pRL-TK质粒共转染到293T细胞中,结合双荧光素酶报告基因检测系统分析不同截短片段的相对荧光活性。利用在线预测软件分析和筛选CCL19基因核心启动子区域内的转录因子结合位点。采用定点突变技术构建转录因子结合位点缺失的荧光素酶报告载体,与pRL-TK质粒共转染到293T细胞,分析转录因子结合位点缺失质粒的相对荧光活性。【结果】 成功构建了7个不同长度(pGL3-P、pGL3-P1、pGL3-P2、pGL3-P3、pGL3-P4、pGL3-P5及pGL3-P6)的CCL19基因启动子片段的荧光素酶报告载体;采用双荧光素酶报告基因检测系统鉴定出转录起始位点上游－256/－186 bp为CCL19基因启动子核心启动子区域,表明该区域对CCL19基因转录调控有重要作用。生物信息学分析预测到该区域存在POU5F1(-201/-189 bp)、ZBTB26(-228/-217 bp)、FOXI1(-239/-228 bp)、GLI2(-255/-243 bp)和SP2(-219/-211 bp) 5个转录因子的结合位点,并成功构建了转录因子结合位点缺失的荧光素酶报告载体。双荧光素酶报告基因检测系统分析显示,POU5F1转录因子的结合位点缺失后绵羊CCL19基因转录活性极显著降低(P<0.01),FOXI1、ZBTB26、SP2转录因子结合位点缺失后绵羊CCL19基因转录活性均极显著升高(P<0.01)。【结论】 试验成功构建CCL19基因启动子荧光素酶报告载体,确定CCL19基因启动子的核心启动子区域为转录起始位点上游－256/－186 bp,并鉴定出转录因子POU5F1结合位点可能是CCL19基因转录的重要调控位点,为下一步研究绵羊CCL19基因在先天性免疫、适应性免疫和淋巴细胞迁移等方面的功能提供理论基础。 相似文献
10.
Mayer B Kis Z Kaján G Frenyó LV Hammarström L Kacskovics I 《Veterinary immunology and immunopathology》2004,98(1-2):85-89
In neonatal calves, maternal immunoglobulin (Ig) is transferred into respiratory secretion which contributes to protection against pathogens. The early predominance of IgG1 in respiratory tract secretions is progressively reduced in favor of IgA by age but in the lower, bronchoalveolar system secreted IgG remains the dominant secreted Ig even in adulthood. The trans-epithelial transport of secretory IgA into mucosal secretions is carried out by the polymeric Ig receptor. However, the mechanism by which IgG crosses epithelial cells to provide defense on mucosal surfaces is still unknown. In order to investigate the possibility that the neonatal Fc receptor, FcRn is involved in this transport we have first analyzed the localization of this receptor in the upper and lower respiratory tracts. Consistent with the in situ hybridization data, immunohistochemistry showed undetectable expression in the tracheal epithelial cells, relatively weak expression in epithelial cells of the bronchi, apparent staining those lining the bronchioli and randomly scattered signal over the alveolar tissue. The bovine FcRn may thus play a role in IgG transport across mucosal epithelial barriers as a trafficking receptor and ensure IgG predominance in the lower respiratory tract. 相似文献
11.
本研究旨在对猪SEPW1基因的潜在启动子区进行克隆及转录活性分析，获得其核心启动子区域，并进一步分析转录因子SP1对SEPW1基因转录活性的影响，为探索SEPW1基因在猪肉质性状方面的功能奠定基础。利用实时荧光定量PCR检测SEPW1基因在大白猪各组织中的表达量，构建空间表达谱；通过PCR技术克隆得到6个逐级缺失的SEPW1基因启动子片段，构建6个双荧光素酶报告载体，通过检测各载体的双荧光素酶活性获得SEPW1基因的核心启动子区域；对核心启动子区进行生物信息学分析，发现潜在的SP1转录因子结合位点；通过过表达、抑制表达、定点突变及凝胶迁移试验（EMSA）确认SP1转录因子结合位点的存在及其对SEPW1基因转录活性的影响。结果显示，SEPW1基因在所检测的4月龄大白猪12个组织中均有表达，其中在腓肠肌及心脏中的表达量较高。双荧光素酶活性显示，猪SEPW1基因5'侧翼区-443~-231 bp为其核心启动子区，且-378~-306 bp存在1个潜在的SP1结合位点。过表达和抑制表达SP1基因结果显示，转录因子SP1能够促进SEPW1基因的转录；定点突变及EMSA试验确认，转录因子SP1可直接与SEPW1基因启动子区的SP1结合位点（-348~-339 bp）相结合。综合以上结果表明，转录因子SP1可直接靶向SEPW1基因的启动子区并促进SEPW1基因的转录。 相似文献
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13.
本研究旨在克隆牛干扰素调节因子9（bovine interferon regulatory factor 9，BoIRF9）基因，分析其生物学信息，并表达其抗原优势区。从感染水疱性口炎病毒（vesicular stomatitis virus，VSV）的EBK细胞中提取总RNA，以反转录得到的cDNA第一条链为模板，参考GenBank中牛IRF9基因序列设计特异性引物进行BoIRF9基因的扩增，对克隆得到的BoIRF9基因进行分子特征、高级结构与分子进化等生物学分析。根据BoIRF9的亲水性、抗原指数和表面可及性，设计1对特异性引物克隆BoIRF9的抗原优势区域片段，将其连接至pET30a（+）载体，构建重组表达质粒pET30a-IRF9-Part。将鉴定正确的质粒转化大肠杆菌Rosetta感受态细胞，经IPTG诱导表达抗原优势区域蛋白，运用SDS-PAGE和Western blotting方法进行蛋白质鉴定。结果显示，本研究克隆得到BoIRF9基因全长1 684 bp，CDS区长1 320 bp，编码439个氨基酸；BoIRF9氨基酸序列与人、马、猪、山羊相似性为80.0%~96.2%；进化树分析结果显示，荷斯坦奶牛与山羊亲缘关系最近，其次为猪、马、非洲象和人；BoIRF9蛋白的分子式为C2139H3342N594O658S18，分子质量为48.5 ku，理论等电点（pI）为5.71；BoIRF9基因编码蛋白不存在跨膜结构，无信号肽，存在1个潜在的N-糖基化位点和多个磷酸化位点；BoIRF9蛋白二级结构含有115个α-螺旋、22个β-转角、68个延伸链和234个无规则卷曲；BoIRF9蛋白包含2个保守结构域，选择包含第一个保守结构域的第12－221位氨基酸作为抗原优势区域，克隆出648 bp的基因片段，并表达出分子质量为27 ku的重组蛋白。本研究结果可为干扰素调节因子的深入研究提供参考。 相似文献
14.
WNT5A(Wnt family member 5A)参与了多种细胞的增殖、凋亡和分化等生物学过程,在乳腺形态发生、毛囊发育等方面发挥了重要作用。该试验利用RT-PCR获得绵羊WNT5A基因的CDS区,分析WNT5A蛋白的结构特征并利用RT-qPCR检测WNT5A基因在9个组织中的表达情况。绵羊WNT5A基因的CDS区全长1062 bp,编码353个氨基酸。该蛋白的二级结构主要以α-螺旋和无规则卷曲为主,β转角、延伸链贯穿于整个蛋白质结构中。String分析结果表明,WNT5A与卷曲蛋白受体、低密度脂蛋白相关蛋白和受体酪氨酸激酶有相互作用。RT-qPCR研究结果表明,WNT5A基因表达具有组织特异性及品种特异性。WNT5A基因在绵羊心脏、肝脏、肺脏、脾脏、肾脏、卵巢、背最长肌、乳腺和皮肤9个组织中均广泛表达,其中在皮肤中的表达量高于其他组织(P<0.05),而在脾脏中表达量较低。它在甘肃高山细毛羊皮肤、乳腺、背最长肌、肾脏、肺脏、脾脏和肝脏中的表达量高于小尾寒羊(P<0.05),而在甘肃高山细毛羊卵巢中的表达量低于小尾寒羊(P<0.05),在心脏中该基因的表达量无差异(P>0.05)。该结果为深入研究绵羊WNT5A基因的功能提供了重要依据,同时也丰富了绵羊基因组内容。 相似文献
16.
本试验旨在进行关岭牛肌球蛋白重链1（myosin heavy chain 1,MYH1）5'侧翼启动子的克隆和生物信息学分析。采集关岭牛血液及组织样品（背最长肌、后腿肌、心脏、肝脏、小肠、脂肪组织）,利用PCR方法扩增关岭牛MYH1基因5'侧翼区,构建关岭牛pUCm-T-MYH1克隆载体,并对MYH1基因5'侧翼区进行生物信息学分析,最后利用实时荧光定量PCR法测定了MYH1基因在关岭牛不同组织中的表达。结果显示,本试验成功获得1 373 bp（-1 360~+12 bp）的MYH1基因5'侧翼启动子序列;利用生物信息软件对获得的克隆序列进行分析发现,MYH1基因5'侧翼启动子序列存在5处可能的转录起始点和多个潜在转录因子结合位点;关岭牛与金丝猴、野猪、家鼠、藏羚羊、野驴的MYH1基因5'侧翼区保守性较强的区域为转录起始点上游-400~+100 bp,推测转录起始点上游-400~+75 bp可能是其核心启动子区域。实时荧光定量PCR分析发现,MYH1基因在关岭牛背最长肌和后腿肌中高表达,在心脏、肝脏、小肠、脂肪组织中表达量很低,说明MYH1基因表达具有组织特异性。 相似文献
17.
牛GDF5基因启动子的克隆与序列分析 总被引：4，自引：1，他引：3
旨在了解生长分化因子5(GDF5)可能的调控序列。本研究通过基因组比对,扩增了牛GDF5基因5′侧翼区,并通过产物纯化、连接、转化及测序比对,确定了2043bp的启动子序列。同时综合考虑已经证实的人GDF5基因启动子结构及应用启动子在线分析软件,对该序列进行分析。结果发现,牛GDF5基因5′侧翼区没有CpG岛,序列比对发现,牛和人GDF5基因启动子区域同源性为78%;牛GDF5基因启动子没有TATAbox或CAATbox结构,其转录起始位点位于翻译起始密码子ATG上游-359bp位置,其潜在的转录因子有AML-la,Ap-1,AmL-la,CdxA,SRY,CdxA,TATA,AmL-la,GTATA-1,MZF1,CdxA,Nkx-2,CdxA,S8和SRY,其中Ap-1,AmL-la,SRY,Nkx-2,S8和SRY高度保守,与人的序列完全一致;推测发现牛GDF5基因启动子-457至-423bp序列中的GT重复序列可以增强启动子的活性,且最小增强子处于-458和-377bp之间。研究结果推测判定了牛GDF5基因启动子的转录起始位置、转录结合位点、活性序列及最小增强子序列,为以后深入研究GDF5基因在牛软骨细胞中的表达机制提供了理论基础。 相似文献
18.
Our understanding of the basis to immunoglobulin formation in cattle has benefited substantially from the application of molecular biology over the past decade. It is now established that both the lambda light chain and heavy chain repertoires are founded upon the frequent expression of single gene families and subgroups of segments which are of conserved sequence. It is likely that a functional kappa locus exists in the bovine genome but this isotype comprises as few as 5% of bovine light chains. Similarly, alternative but non-expressed V(H) gene families are present posing intriguing but unresolved questions about the regulation of immunoglobulin synthesis. The heavy chain frequently bears a third complementarity-determining region which is atypically long but the processes which expand this region of the reading frame and its contribution to the interaction with antigen remain matters of speculation. Opportunities exist to map the major immunoglobulin loci and to define the membership and sequence diversity of the gene families which dominate each repertoire. However, it is already evident that cattle cannot generate significant diversity from rearrangement and junctional imprecision alone. Elucidation of the mechanism(s), dynamics and tissue distribution of immunoglobulin diversification in cattle, thus, remain key challenges in this branch of veterinary immunology. 相似文献
19.
本试验先后进行2轮载体构建及转染试验,对牛乳铁蛋白基因启动子元件调控效应进行了分析。首先进行第1轮载体构建及转染试验：采用PCR方法,利用4对引物从牛乳铁蛋白基因5′调控区-1 799向3′端依次缺失500bp进行调控序列扩增,将获得的扩增片段替换pEGFP-N1的CMV启动子,构建了4个重组载体,将重组载体转染牛乳腺上皮细胞（BMEC）,经筛选获得稳定的单克隆细胞,测定并比较各组细胞的荧光强度。在以上试验基础上,为进一步精确确定牛乳铁蛋白基因启动子顺式调控元件序列,进行第2轮载体构建及转染试验,方法同第1轮。结果表明,牛乳铁蛋白基因上游调控序列-1 323～-1 372存在负调控元件,-1 372～-1 560存在正调控元件。 相似文献
20.
旨在克隆牛HSL基因的CDS序列,并对该基因进行生物信息学、组织表达谱分析。根据GenBank登录的牛HSL基因序列(NM_001080220)设计1对引物,对牛组织样品中的HSL基因CDS序列进行RT-PCR扩增及序列测定;利用半定量RT-PCR方法检测HSL基因在牛各组织中的表达情况;利用生物信息学软件对其所编码的牛HSL蛋白进行分析。结果显示,获得的牛HSL基因CDS全长为2271bp,编码756个氨基酸,与GenBank登录的牛HSL基因同源性最高,为99.9%。HSL蛋白含有一个HSL-Nsuperfamily保守结构域,无信号肽结构,具有疏水性。半定量RT-PCR显示,HSL基因在检测的心脏、脾脏、肺脏、肾脏、肝脏、大网膜、皮下脂肪、肌肉8种组织中均有表达,其中在大网膜和皮下脂肪中表达量较高,在肾脏、脾脏、肝脏、肺脏、肌肉和心脏中度表达。该试验可为研究牛HSL蛋白的结构和功能提供参考。 相似文献