湖羊Lrh-1基因cDNA序列及组织表达谱分析

本研究以湖羊肝脏为材料对肝受体类似物-1(Liver receptor homolog-1,Lrh-1;NR5A2)基因序列进行RT-PCR和RACE测定,并用DNAman、Tmpred、Signal P 3.0、Expasy等生物信息学分析软件和在线工具,对Lrh-1cDNA序列及其蛋白的理化特性、跨膜结构、信号肽和二级结构进行生物信息学分析,同时采用实时荧光定量PCR方法检测湖羊Lrh-1基因的组织表达谱。结果表明,湖羊Lrh-1基因cDNA序列全长1 488bp,与牛的核酸序列相似度最高,为98%,编码区共编码495个氨基酸,氨基酸序列与牛、人、马、猴、犬、小鼠、褐鼠的氨基酸同源性分别为98%、97%、96%、97%、97%、86%和86%;Lrh-1mRNA在湖羊脑、消化及生殖系统组织中均有表达且具有组织特异性,其中在下丘脑组织中的表达量相对最高。     

牦牛SLC25A31基因的CDS序列及其表达蛋白生物信息学分析

为了研究牦牛ANT4在脑组织中的表达情况,试验通过RT-PCR技术克隆牦牛SLC25A31基因的cDNA序列,并利用DNAMAN、MAGA4、SWISS-MODEL、ExPASy等生物信息学软件系统对其进行分析。结果表明:扩增出的牦牛SLC25A31基因的编码区长972 bp,编码323个氨基酸;与普通牛、鼠和人相应基因核苷酸序列进行比对,序列一致性分别为99.28%、84.88%和89.81%;其编码蛋白分子质量为35.695 ku,含多个修饰位点。说明SLC25A31基因的CDS序列及其表达蛋白的结构在高原牦牛与普通牛间并无明显差异,保守性极强;并且通常在睾丸中表达的ANT4在高原牦牛脑组织中也能表达。     

猪CTSD全长cDNA的克隆和表达分析

以人CTSD mRNA全长序列为基序,在db EST库中搜索同源性大于80%、重叠大于40个碱基的猪EST下载经Seqman软件装配后,利用RT-PCR扩增到猪CTSD基因部分cDNA序列,进一步通过RACE技术获得cDNA全长（GenBank登录号为DQ018727）,猪CTSD基因cDNA全长2032 bp包括93 bp 5′UTR区、706 bp 3′UTR区和1 233bp ORF,编码410个氨基酸残基。其编码区与人、鼠、牛、羊CTSD编码区同源性分别为87.9%、78.2%、86.6%和85.0%,其氨基酸序列与人、鼠、牛、羊氨基酸序列同源性分别为86.6%、80.1%、86.0%和84.7%。疏水性分析和信号肽预测发现猪CTSD氨基酸序列存在至少7个疏水性区域,信号肽位点为第1-20个氨基酸残基。在NCBI进行Blast P搜索结果显示猪CTSD氨基酸结构中含有Asn（Asparagine,天门冬酰胺）结构域,与天冬氨酸蛋白酶3D结构同源性高达99.7%,具有真核生物天冬氨酸蛋白酶的典型结构。以猪多组织RNA池为模板,以1026 bp部分cDNA序列作为杂交探针进行Northern杂交,得到一条2000 bp左右特异条带,从而验证了RACE产物为全长cDNA并揭示该基因只有一个转录本。为了研究猪CTSD基因在体内不同组织内表达的特点,采用半定量RT-PCR对CTSD基因在猪10个组织中的表达进行研究,结果表明在10个组织中均有表达,且表达量与-βactin内参接近。     

猪CD163基因的克隆及截短原核表达

（目的）研究旨在克隆猪CD163 (pCD163)基因、体外表达CD163蛋白。（方法）采用RT-PCR扩增猪CD163基因，运用生物信息学软件分析其核苷酸和编码氨基酸序列，将猪CD163的两个截短基因片段（1-1749 bp和1717-3348 bp）克隆至原核表达载体pGEX-4T-1，构建重组原核表达载体，转化Transetta(DE3)菌株，IPTG诱导蛋白表达，采用SDS-PAGE和Western blot进行蛋白鉴定。（结果）结果表明，pCD163开放阅读框为3348 bp(GenBank:OM321546)，编码1116 aa，蛋白分子量为120.5 kDa，该基因与已经测定的人、绿猴、牛、狼的CD163的核苷酸相似性依次为87.8%、88%、87.4%和88%,pCD163基因与抹香鲸和牛的CD163基因亲缘关系最近，与人、狼和绿猴的亲缘关系相对较近；pCD163截短片段1-1749蛋白分子量为92 kDa，截短片段1717-3348蛋白分子量为85 kDa，均以包涵体形式表达。（结论）试验成功克隆了pCD163基因，两个截短基因片段在原核细胞中均以包涵体形式表达，为后...     

荣昌猪白细胞介素-2受体γ链基因克隆、表达与鉴定

运用RT-PCR技术从由刀豆蛋白(ConA)诱导培养的荣昌猪外周血单核淋巴细胞(PBMC)扩增出猪白细胞介素-2受体γ链基因的完整开放阅读框(ORF),长约1 107 bp,编码由368个氨基酸残基组成的相对分子质量为41 780的蛋白多肽.荣昌猪IL-2Rγ与人、猕猴、牛、犬、鼠在氨基酸水平上的同源性分别为81.6%,80.8%,85.1%,83.2%和68.8%.运用PCR技术从含荣昌猪IL-2Rγ,开放阅读框序列质粒中扩增其成熟蛋白编码基因,共1 020 bp.将其定向克隆于原核表达载体pET-32a(+)后在E.coli BL21中诱导表达,SDS-PAGE结果显示表达的融合蛋白约为52 000,重组蛋白以包涵体的形式表达,表达产物约占茵体总蛋白的37.6%;Western-blotting分析表明,在相对分子质量52 000处有一奈特异性的带;对γ诱导重组茵进行转录检测得到约1 020 bp的特异性片段.     

牛源IL-6基因克隆与生物信息学分析

试验旨在克隆牛白细胞介素-6（IL-6）基因，并对其编码蛋白进行生物信息学分析，对荷斯坦奶牛进行尾根采血提取RNA后逆转录为cDNA，根据NCBI数据库中已知的牛IL-6基因mRNA序列设计特异性引物，应用RT-PCR技术克隆得到CDS区全长序列，利用生物信息学软件分析牛IL-6基因序列特征、同源性及编码产物的理化性质等。结果表明，试验成功克隆了牛IL-6基因CDS区，全长627 bp，分子质量为23.759 ku，共编码208个氨基酸，理论等电点为7.58，脂溶系数为93.37，属于亲水性蛋白；牛IL-6基因与猪、绵羊、大鼠、人、猫、犬、鸡、小鼠、马和兔的同源性分别为84.4%、96.2%、66.7%、77.5%、75.9%、79.3%、48.5%、67.0%、79.5%和67.5%；系统进化树分析发现，牛与绵羊亲缘关系最近，猪次之，鸡最远；在二级结构预测中，该蛋白无规则卷曲与α-螺旋分别为34.6%和60.1%；亚细胞定位于细胞质；该蛋白含有信号肽和跨膜螺旋结构。本研究为进一步分析牛IL-6基因的功能奠定了一定的理论基础。     

鹿茸X型胶原基因的全长cDNA克隆及序列分析

采用RT-PCR和RACE技术,从鹿茸软骨细胞总RNA中扩增克隆了梅花鹿X型胶原（collagen X,colX）全长cDNA序列,并进行了序列特性分析。序列分析结果表明,梅花鹿colX全长cDNA序列为3135nt,其中5′非编码区96nt、3′非编码区1014nt和2022nt的开放阅读框编码674个氨基酸的鹿colX前体蛋白,与牛、猪、犬、人类和小鼠等脊椎动物colX氨基酸序列之间的同源性超过82%。按该基因氨基酸序列构建了进化树,揭示其与牛基因更加同源。     

藏獒犬白介素18基因的克隆及原核表达

通过RT-PCR技术从刺激的藏獒犬外周血淋巴细胞中成功克隆白介素18基因全长,其大小为582bp,编码194个氨基酸。与GenBank上发表的犬IL-18(NM001003305)比较,序列的同源性为100%,与犬、猫、猪,牛、羊、人的IL-18基因核苷酸同源性分别为100%、91.2%、91.1%、88.5%、89.3%、84.4%。系统进化树可以看出,藏獒犬基因与犬的亲缘关系最近,与猫的其次,与人是较远的。然后构建-IL18去信号肽的原核表达载体pET-30a-IL18,转化BL2L,IPTG诱导,表达产物经SDS-PAGE分析表明,表达出25kd融合蛋白而且目的蛋白主要以包涵体的形式存在,westbloting验证表达的蛋白正确。     

猪CD58分子基因克隆、表达及其结构功能预测

CD58在机体免疫系统中具有重要作用,本研究通过对人、绵羊CD58 mRNA序列比对,设计兼并引物,应用反转录PCR技术克隆猪CD58基因,并在大肠杆菌中进行原核表达,同时运用生物信息学方法对其核苷酸序列、编码的氨基酸序列以及蛋白结构进行预测。结果表明:克隆的猪CD58 cDNA全长800 bp,ORF为735 bp;将其在大肠杆菌中进行表达,产物可被CD58抗血清识别;序列比对结果显示猪、羊和人的CD58核苷酸序列及氨基酸序列同源性并不高,但蛋白结构预测表明三者蛋白结构非常相似,尤其是V区三维结构,这是异种动物淋巴细胞和红细胞发生黏附的分子基础。该研究为CD58作为疫苗佐剂或免疫调节药物在临床中的应用、进一步研究CD58分子结构及CD2-CD58复合物激活免疫系统的机理奠定了基础。     

猪DECR1基因cDNA的克隆、序列分析及原核表达研究

旨在研究猪2,4-dienoyl-CoA reductase1(DECR1)基因的结构,揭示该基因的原核表达规律。试验以山西马身猪的肝脏组织为材料,采用RT-PCR与RACE技术克隆了DECR1基因的cDNA全序列,并将其重组于pET32a+原核表达载体中,经酶切、序列鉴定正确后,重组质粒转化大肠杆菌BL21进行诱导表达。结果表明:猪DECR1基因的cDNA全长2352bp,包括987bp的开放阅读框(ORF),53bp的5′非翻译区(UTR)和1312bp的3′-UTR;编码区(CDS)编码328个氨基酸残基与猪(电子预测序列)、牛、人、猩猩、猴、马、犬、鼠相应序列的同源性分别为99%、88%、88%、87%、87%、87%、87%和83%;SDS-PAGE电泳结果显示,在IPTG诱导4h时,外源蛋白表达效率最高;Western blot检测发现经诱导表达的蛋白产物大小约为35ku,与预测的大小一致。猪DECR1基因的克隆和表达研究,为进一步探究该基因的生物学功能及其分子遗传机制提供了理论基础。     

Cloning of bovine CD34 cDNA

CD34 is a leukocyte antigen that is expressed in various cell types including hematopoietic cells. Monoclonal antibodies against human, murine, and canine CD34 proteins have been used for the identification of lymphohematopoietic stem/progenitor cells. The cDNA encoding bovine CD34 was cloned, and its nucleotide sequence was determined. The identity of the deduced amino acid sequence of the encoded protein to those of human, murine. and canine CD34 proteins was 61.1%, 56.0%, and 66.1%, respectively. Northern blot hybridization with the cDNA as a probe detected CD34 RNA expression in the cerebrum, spleen, heart, and lung of a fetal calf.     

CD34 protein is expressed in murine,canine, and porcine lungs

The cell surface protein CD34 is expressed in various human tissues and cells, including hematopoietic stem cells, vascular endothelial cells, mucosal dendritic cells, mast cells, eosinophils, microglia, fibrocytes, muscle satellite cells, and platelets. There is a lack of data on the expression of CD34 in canine and porcine tissues. Therefore, we designed a series of immunoblotting, immunohistochemistry, and immunofluorescence experiments to observe CD34 expression in murine, canine, and porcine lungs. We used a rabbit antibody (clone EP373Y) to target the conserved human CD34 C-terminal region and validated its immunoreactivity against mouse lung homogenates. The data showed diffuse bronchiolar and alveolar epithelial localization of CD34 protein in normal murine, canine, and porcine lungs. At 9 or 24 h after bacterial endotoxin exposure, murine CD34 protein shifted to specific bronchoalveolar cells with a punctate pattern, as quantified by CD34 fluorescence. Specific porcine bronchoalveolar cells and leukocytes had significant CD34-positive immunostaining after H3N1 influenza infection. Thus, our study provides fundamental data on the expression of CD34 in lungs and validates an antibody for use in further experiments in these animal species.     

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (γc) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34⁺ bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting ∼75% of Cf2Th cells. An average of 11% of highly enriched canine CD34⁺ cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine γc cDNA produced a γc protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34⁺ BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible. Suter, S.E., Gouthro, T.A., McSweeney, P.A., Nash, R.A., Haskins, M.E., Felsburg, P.J. and P.S. Henthorn, 2006. Optimized transduction of canine paediatric CD34⁺ cells using an MSCV-based bicistronic vector. Veterinary Research Communications, 30(8), 881–901     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

Characterization of CD34+ cells from canine umbilical cord blood, bone marrow leukocytes, and peripheral blood by flow cytometric analysis

Characterization of CD34+ cells in canine bone marrow, umbilical cord blood, and peripheral blood was performed by flow cytometric analysis. The ratio of CD34+CD45hi cells, which are absent in human blood, was high in the CD34+ cell fraction, but 98% of these was suggested B-cells. The remaining CD34+CD45lo cells may comprise canine hematopoietic progenitor cells, and these cells accounted for 0.23 +/- 0.07% of the fraction in cord blood, 0.30 +/- 0.07% in bone marrow, and 0.02 +/- 0.01% in peripheral blood.     

Cloning and sequencing full length of canine Brca2 and Rad51 cDNA.

Mammary tumors are the most common neoplasm in female dogs, Canis canis, and in women. Mutations in human Brca2 confer an increased risk of female breast cancer. Previous studies have shown that the Brca2 tumor suppressor protein interacts with the recombinational repair protein Rad51. We cloned the full-length cDNA of the canine homologues of Brca2 and Rad51 to obtain a basis for studying their relationship with susceptibility to mammary tumors. The canine Brca2 and Rad51 cDNAs are 11 and 1.5 kb long, encoding 3.471 and 339 amino acids, respectively. The amino acid sequence of canine Brca2 showed 68% homology with the human protein, and 58% homology with a murine protein. There were highly conserved regions in the C-terminus of all three proteins, where the Rad51 interacting domain and putative nuclear localization signals are located. Comparing with the partial genomic sequence previously reported, we found possible nuclear polymorphisms in exon 11, some of which result in amino acid substitutions. On the other hand, canine Rad51 protein had extremely high homology (99%) to the human and murine proteins. Expression of both Brca2 and Rad51 was detected in the mammary gland, suggesting that these two genes interact in the canine mammary gland.     

A functional comparison of canine and murine bone marrow derived cultured mast cells

Disorders involving mast cells are extremely common in dogs, ranging from allergic diseases to neoplastic transformation resulting in malignant mast cell tumors. Relatively little is known regarding the basic biologic properties of normal canine mast cells, largely due to the difficulty in reliably purifying large numbers from canine skin. In vitro generated bone marrow derived cultured mast cells (BMCMCs) are routinely used in both human and murine studies as a ready source of material for in vitro and in vivo studies. We previously developed a technique to generate canine BMCMCs from bone marrow derived CD34+ cells and demonstrated that these cells exhibit the phenotypic properties characteristic of mast cells and release histamine in response to IgE cross-linking. The purpose of the following study was to characterize the functional properties of these canine BMCMCs and contrast these with the functional properties of murine BMCMCs. Our work demonstrates that both IL-4 and IL-10 promote canine BMCMC proliferation, possibly through upregulation of Kit expression, while TGFbeta inhibits proliferation. The canine BMCMCs produce a variety of cytokines and chemokines in response to IgE cross-linking and chemical stimulation including IL-3, IL-4, IL-13, GM-CSF, RANTES, and MIP1alpha. Interestingly, the canine BMCMCs released significantly larger amounts of MCP-1 and tryptase and significantly smaller amounts of IL-6 following chemical stimulation and IgE cross-linking when compared to murine BMCMCs. Lastly, the canine BMCMCs produced larger amounts of active MMP9 than their murine counterparts. In summary, canine BMCMCs exhibit unique functional properties that distinguish them from murine BMCMCs and provide insight into the contribution of these cells to mast cell disorders in the dog.     

Isolation and characterization of pediatric canine bone marrow CD34+ cells

Historically, the dog has been a valuable model for bone marrow transplantation studies, with many of the advances achieved in the dog being directly transferable to human clinical bone marrow transplantation protocols. In addition, dogs are also a source of many well-characterized homologues of human genetic diseases, making them an ideal large animal model in which to evaluate gene therapy protocols. It is generally accepted that progenitor cells for many human hematopoietic cell lineages reside in the CD34+ fraction of cells from bone marrow, cord blood, or peripheral blood. In addition, CD34+ cells are the current targets for human gene therapy of diseases involving the hematopoietic system. In this study, we have isolated and characterized highly enriched populations of canine CD34+ cells isolated from dogs 1 week to 3 months of age. Bone marrow isolated from 2- to 3-week-old dogs contained up to 18% CD34+ cells and this high percentage dropped sharply with age. In in vitro 6-day liquid suspension cultures, CD34+ cells harvested from 3-week-old dogs expanded almost two times more than those from 3-month-old dogs and the cells from younger dogs were also more responsive to human Flt-3 ligand (Flt3L). In culture, the percent and number of CD34+ cells from both ages of dogs dropped sharply between 2 and 4 days, although the number of CD34+ cells at day 6 of culture was higher for cells harvested from the younger dogs. CD34+ cells harvested from both ages of dogs had similar enrichment and depletion values in CFU-GM methylcellulose assays. Canine CD34+/Rho123lo cells expressed c-kit mRNA while the CD34+/Rhohi cells did not. When transplanted to a sub-lethally irradiated recipient, CD34+ cells from 1- to 3-week-old dogs gave rise to both myeloid and lymphoid lineages in the periphery. This study demonstrates that canine CD34+ bone marrow cells have similar in vitro and in vivo characteristics as human CD34+ cells. In addition, ontogeny-related functional differences reported for human CD34+ cells appear to exist in the dog as well, suggesting pediatric CD34+ cells may be better targets for gene transfer than adult bone marrow. The demonstration of similarities between canine and human CD34+ cells enhances the dog as a large, preclinical model to evaluate strategies for improving bone marrow transplantation protocols, for gene therapy protocols that target CD34+ cells, and to study the engraftment potential of various cell populations that may contain hematopoietic progenitor cell activity.     

Molecular cloning of canine mucosal addressin cell adhesion molecule-1 cDNA and its expression in normal tissues

A cDNA encoding canine mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was cloned. The entire open reading frame of canine MAdCAM-1 cDNA comprises 1137 bp, corresponding to 378 amino acid residues. The deduced amino acid sequence of canine MAdCAM-1 was 55.2%, 53.7%, and 52.4% identical to rat, mouse, and human MAdCAM-1, respectively. Canine MAdCAM-1 appeared to contain two immunoglobulin-like domains at the N-terminus, followed by a mucin-like domain and a third immunoglobulin-like domain. The structures of the dog, rat, and mouse proteins are likely similar because all of the cysteine residues in the immunoglobulin-like domains were conserved. Canine MAdCAM-1 mRNA was confirmed to express extremely in the mesenteric lymph node by RT-PCR.