Molecular cloning and expression analysis of pig CD79alpha

The CD79alpha (immunoglobulin alpha, Igalpha), a part of B cell receptor (BCR) complex, forms a heterodimer with CD79beta (Igbeta) and plays an important role in the B cell signaling. In this study, we have cloned pig Cd79a cDNA using RT-PCR and determined the complete cDNA sequence of pig Cd79a. Pig Cd79a cDNA contains an open reading frame (672bp) encoding 223 amino acids. The putative amino acid identity of pig CD79alpha with those of human, cattle and mouse are 70.4, 81.4, and 67.7%, respectively. Alignment of the CD79alpha amino acid sequence with those of mammalian species showed that the extracellular domain is the most divergent, whereas transmembrane region and cytoplasmic tail including immunoreceptor tyrosine-based activation motif (ITAM) are largely conserved. Pig Cd79a mRNA was detected mainly in lymphoid tissues by RT-PCR. The highest level of Cd79a mRNA expression was observed in mesenteric lymph node and spleen. Relatively low level of Cd79a mRNA expression was observed in lung, thymus and small intestine. The lowest level of Cd79a mRNA expression was observed in large intestine. Flow cytometry analyses demonstrated that human CD79alpha antibody recognizes a CD79alpha in pig B cells. Further, immunohistochemistry analysis using human CD79alpha antibody on pig spleen was revealed that CD79alpha is strongly expressed in the follicular mantle zone rather than in the germinal center. Future study will be focused on defining the functional role of CD79alpha during the course of pig infectious diseases and the formation of neoplasm.     

Molecular cloning and structural analysis of the porcine homologue to CD97 antigen

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.     

Molecular cloning and expression analysis of pig CD7

CD7 is an integral membrane protein which mediates an important signal to mediate the differentiation, activation, and regulation of some T cells and NK cells. However, only human and mouse CD7 have been identified and studied among mammalian species. In this study, we cloned pig CD7 cDNA and determined its complete cDNA sequence. Pig CD7 cDNA contained an open reading frame (627 bp) encoding 208 amino acids with well conserved motifs involved in signal transduction within cytoplasmic tail among mammalian species. Pig CD7 mRNA was detected by RT-PCR in mainly lymphoid tissues, indicating the conserved functions of CD7 in pigs. Moreover, we generated soluble pig CD7 fusion immunoglobulin (pig CD7Ig) containing extracellular domain of pig CD7 to test whether pig CD7 binds to pig galectin-3. Flow cytometry and immunohistochemistry analyses indicated that soluble pig CD7Ig can bind to galectin-3 expressed in macrophages and epithelial cells of small intestine. These results help to analyze the structural relationship between CD7 and its ligand transferring signal transduction among mammalian species.     

牛Fas基因的组织表达分析及其功能预测

采用RT-PCR技术从中国西门塔尔牛睾丸组织总RNA中反转录FascDNA,将其克隆于pMD19-Tvector后进行测序分析,并对其表达的蛋白结构功能进行预测、分析。结果表明:牛的FascDNA序列为1109bp,编码323个氨基酸残基,在氨基酸序列上与羊、猪、人、小鼠的相似性分别为90.5%、65.3%、56.7%和48.6%。Fas蛋白的胞外区有2个糖基化位点和3个富含半胱氨酸残基的结构亚域,对Fas蛋白的定位及凋亡信号识别有重要作用。牛与羊、猪、人、小鼠Fas的死亡结构域(Death domain,DD)氨基酸序列的相似性分别为94.3%、68.5%、59.6%和70.8%,体现了该结构在各物种间较强的保守性及接受凋亡信号诱导靶细胞凋亡的重要性。组织表达谱发现,Fas不仅表达于牛的淋巴、脾等淋巴系统,而且高表达于牛生殖系统的睾丸中,这对于进一步阐明Fas在公牛精子发生过程中的调控作用奠定基础。     

山羊组织蛋白酶L基因克隆、序列分析及组织表达研究

试验旨在克隆山羊组织蛋白酶L(cathepsin L,CTSL)基因,并进一步探讨该基因结构与功能的关系,揭示其组织表达规律。本研究以天府肉羊为试验材料,运用同源序列克隆技术,对山羊CTSL基因进行克隆,并对其进行生物信息学分析及组织表达研究。结果表明,山羊CTSL基因编码区(CDS区)长1005 bp,共编码334个氨基酸;山羊CTSL氨基酸与绵羊的同源性最高(99.10%),其次是牛(96.71%)、猪(90.12%)、人(76.95%)、大鼠(76.12%)、小鼠(75.82%)、青鳉鱼(66.17%)和斑马鱼(61.42%);经预测山羊CTSL可能含有1个Inhibitor_129结构域和1个Pept_C1结构域;山羊CTSL基因在11个组织中均有表达,但在肺脏和肾脏中的表达量显著高于在心肌、肝脏、脾脏、胸肌、腹肌、腿肌、膈肌、眼肌和比目鱼肌中的表达量（P<0.05）。     

猪ICOS基因部分cDNA的克隆与序列分析

研究旨在对猪T细胞诱导型刺激物(ICOS)基因的cDNA序列进行克隆与分析。根据已报道的人ICOS基因cDNA序列设计引物,首次从猪脾脏组织总RNA中扩增出ICOS基因编码区全长cDNA序列,克隆于pMD18-T载体后进行测序并进行序列拼接,运用生物信息学分析DNA序列。结果表明:该基因编码区全长630bp,编码210个氨基酸,包含5个外显子。该序列与人全基因核苷酸序列及推导的氨基酸序列的同源性分别为80%和85%;与狗和小鼠的推导氨基酸序列的同源性分别为81%和75%。这为进一步研究该基因的结构特点和功能奠定了良好基础。     

西农萨能奶山羊SERPINA1基因克隆、生物信息学及组织表达分析

本研究旨在克隆西农萨能奶山羊SERPINA1基因的CDS区,采用生物软件和在线预测工具进行生物信息学分析,通过实时荧光定量PCR技术检测SERPINA1基因在西农萨能奶山羊各组织间mRNA的表达水平。根据GenBank中山羊SERPINA1基因CDS区序列（登录号：XM_018066209.1）,利用Primer Premier 5.0软件设计特异性引物,RT-PCR扩增目的基因,构建原核表达载体测序后对序列进行生物信息学分析;采集西农萨能奶山羊心脏、肝脏、脾脏、肺脏、乳腺、肾脏、肌肉、瘤胃和小肠组织,提取组织RNA,反转录为cDNA模板,设计特异性定量引物,进行实时荧光定量PCR,检测SERPINA1基因在不同组织中的表达差异。结果显示,西农萨能奶山羊SERPINA1基因CDS区全长1 326 bp,编码441个氨基酸;同源性比对分析显示,西农萨能奶山羊与山羊、绵羊、牛和小鼠SERPINA1基因核苷酸序列同源性分别为100%、98.6%、95.7%和71.6%,与山羊亲缘关系最近,其次是绵羊。SERPINA1蛋白分子质量为48.71 ku,等电点为5.71,为跨膜亲水蛋白;SERPINA1氨基酸序列分别有62个磷酸化位点,3个跨膜区结构。组织表达分析显示,SERPINA1基因在西农萨能奶山羊肝脏组织中显著高表达（P<0.05）,其次是乳腺组织,在肺脏组织中表达量最低。研究结果为进一步探究SERPINA1基因在奶山羊乳蛋白合成代谢中的作用提供理论依据。     

Canine Bcl-xL gene and its expression in tumor cell lines

The canine Bcl-xL gene was cloned and sequenced. Canine Bcl-xL cDNA clone was 1252 bp in length, and encoded 233 deduced amino acides. The predicted canine Bcl-xL amino acid sequence shared 99.6%, 97.0%, 97.9%, 98.7% and 98.3% homology with that of human, mouse, rat, sheep and pig Bcl-xL, respectively. RT-PCR analysis revealed that canine Bcl-xL mRNA was constitutively expressed in CL-1 (canine lymphoma) and GL-1 (canine B cell leukemia) cell lines.     

藏山羊SFRS18基因克隆、表达及其与肌内脂肪含量相关性研究

试验旨在克隆藏山羊SFRS18(splicing factor arginine/serine-rich 18)基因CDS序列,并进行生物信息学分析,同时分析其组织表达特征以及与肌内脂肪含量进行相关性分析,为深入研究该基因在山羊肌内脂肪沉积中的作用积累数据。采用RT-PCR技术获得藏山羊SFRS18基因序列,结合生物信息学分析蛋白的理化性质、结构和不同物种的同源性,实时荧光定量检测SFRS18 mRNA表达情况,并将表达量与肌内脂肪含量相关联。结果表明,藏山羊SFRS18基因cDNA序列长为1299 bp,开放阅读框(ORF)长为1272 bp,编码423个氨基酸,蛋白分子结构式为C₂₀₀₃H₃₄₂₄N₇₆₀O₆₉₆S₄,分子质量为49.42 ku,等电点pI=11.20,SFRS18蛋白为不稳定的亲水性蛋白,无信号肽;有103个磷酸化位点,2个N-糖基化位点和39个O-糖基化位点;亚细胞定位于在细胞核(82.6%)、细胞质(8.7%)、细胞骨架(4.3%)和质膜(4.3%),属于非跨膜蛋白;预测二级结构由0.71% α-螺旋和99.29%无规则卷曲组成;藏山羊核苷酸序列和氨基酸序列与牛、绵羊和水牛的相似性最高(99%),系统进化树分析表明藏山羊与牛亲缘关系最近;实时荧光定量PCR结果显示,SFRS18基因在藏山羊的不同组织中都存在表达,其中在脾脏中表达水平最高,在背最长肌中表达水平最低;在藏山羊背最长肌和腿肌中SFRS18 mRNA表达与肌内脂肪含量均呈显著正相关(r=0.081,P<0.05;r=0.373,P<0.05)。SFRS18基因可以作为调节山羊脂肪沉积的候选基因。     

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.     

猪精子发生候选基因PYGO2的分离、表达和亚细胞定位研究

旨在分离猪PYGO2编码区序列，获悉该基因mRNA组织表达模式、蛋白质结构特征、细胞中的分布和定位，并构建蛋白相互作用网络。本研究首先利用RT-PCR从成年版纳微型猪近交系（BMI）睾丸组织中克隆PYGO2编码区序列；利用生物信息学解析其基因结构并对蛋白质进行多种功能分析，比较多个哺乳动物PYGO2的氨基酸序列同源性，构建系统进化树和蛋白质相互作用网络；然后利用qPCR技术检测PYGO2在15个组织中的mRNA表达情况；最后通过构建pEGFP-C1-PYGO2融合表达载体，转染猪睾丸细胞（ST），确定PYGO2的亚细胞定位。结果表明，PYGO2基因CDS长1 221 bp，编码406个氨基酸（基因和氨基酸号分别为KY644518和AVB77243.1），定位在猪4号染色体；PYGO2蛋白二级结构以无规则卷曲为主，N端和C端均疏水；氨基酸序列同源比对分析表明，猪PYGO2与其他哺乳动物的相似度均大于97%，在进化上高度保守；蛋白互作网络分析显示，猪PYGO2与9个蛋白可能存在相互作用，其中与BCL9蛋白作用最为紧密；qPCR表达分析表明，猪PYGO2在被检的15个组织中均有不同程度表达，在生殖腺中表达相对较高；ST细胞中的亚细胞定位结果表明，PYGO2 mRNA主要分布在细胞核。本研究获得了PYGO2基因的编码序列，蛋白质结构和定位，蛋白质相互作用网络，mRNA多组织表达特征，可为进一步解析该基因在猪精子生成中的分子机制提供参考。     

Cloning of Full Length cDNA and Tissue Expression Analysis of Hepcidin Gene from Cobia (Rachycentron canadum)

The techniques of homology clone and RACE were used to clone the Hepcidin gene from cobia (Rachycentron canadium).The full length cDNA of Hepcidin gene was 714 bp with a 213 bp 5'untranslated region (UTR),a 228 bp 3'UTR and a 273 bp open reading frame (ORF) encoding a polypeptide of 90 amino acid residues with a predicted molecular weight of 10.03 ku and theoretical isoelectric point of 7.54.The predicted molecular included signal peptide,prodomain peptide and mature peptide.Phylogenetic tree of Hepcidin amino acid sequences was constructed and homology compapison of amino acid sequences showed that homologies were varied from 24.4% to 85.6% with some known Hepcidin amino acids in other fishes and mammals.Quantitative Real-time PCR (qRT-PCR) analysis revealed that Hepcidin gene was expressed in all tissues with different expression levels,which expressed most in liver.The Hepcidin gene expressions in liver,head kidney and spleen were up-regulated after stimulation of LPS and formalin-inactivated Vibrio carchariae.