关于牛CD34cDNA的克隆及应用

CD34是白细胞抗原，可在不同类型的细胞包括造血细胞中进行表达，抗人，鼠和犬CD34鼠白的单克隆抗体已用于对淋巴造血干细胞的鉴别，本试验克隆出编码牛CD34的cDNA，并测定其核苷酸顺序，推测其蛋白的氨基酸顺序与人，鼠和犬的CD34蛋白的氨基酸顺序的同源性分别为61.1%,56.0%,和66.1%。并以cDNA作为探针进行Northern杂交，探测CD34RNA在胎牛脑，脾，心和肺中的表达。     

Cloning of bovine CD69

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.     

猪CD3ε分子的cDNA 克隆与序列分析

应用RT-PCR和3′RACE技术，本研究从猪外周血淋巴细胞总RNA中扩增克隆了猪CD3ε基因并进行了序列分析。序列分析结果表明，猪CD3ε cDNA序列长1241nt，其中5′非翻译区80nt，3′非翻译区570nt，开放阅读框591nt，该开放阅读框编码196个氨基酸残基的CD3ε前体蛋白(无糖基化位点)；在猪CD3ε蛋白的胞外区，Cys^49、Cys^91、Cys^108和Cys^111为保守性氨基酸残基，它们与CD3ε免疫球蛋白样结构的形成有关。在猪CD3ε蛋白胞浆区，存在免疫受体酪氨酸活化基序(ITAM)、内质网潴留基序和1个SH3结合基序，其中ITAM基序含有2个SH2结合基序和2个潜在酪氨酸磷酸化位点Tyr^177和Tyr^188，这些基序是猪CD3分子参与T细胞活化信号转导和TCR-CD3复合体装配的结构基础。推导氨基酸序列分析结果表明，猪与人、鼠、狗、牛和绵羊CD3ε蛋白的氨基酸同源性分别为61．2％、58．7％、58．7％、65．1％和65．6％。     

猪CD4分子的全长cDNA克隆与序列分析

CD4分子为动物辅助性T细胞（TH）和部分胸腺细胞的共受体与信号传导分子，参与TCR介导的TH细胞活化和胸腺细胞分化过程。本研究应用RT-PCR和RACE技术从猪胸腺细胞总RNA中扩增克隆了猪CD4全长cDNA序列，并进行了序列特性分析。序列分析结果表明，在猪胸腺细胞和外周血淋巴细胞中存在2种方式剪接的CD4 mRNA转录本，其中一种mRNA转录本缺失编码40个氨基酸残基的120nt序列，提示该转录本可能编码猪分泌型CD4分子；猪CD4全长cDNA序列为2715nt。其中5′非编码区159nt，3′非编码区1183nt，1374nt的开放阅读框编码457个氨基酸的猪CD4前体蛋白（含4个糖基化住点）；猪CD4分子的7个Cys残基（Cys^43、Cys^122、Cys^327、Cys^418、Cys^421、Cys^444和Cys^446）和2个Ser残基（Ser^432和Ser^439）在动物种间保守；在猪CD4分子胞浆区．存在高度保守的Src家族蛋白酪氨酸激酶p56^kk。识别位点KKTCQC和内化相关双亮氨酸基序。推导氨基酸序列分析结果显示，猪与人、免、猫、狗和鼠CD4蛋白的氨基酸同源性分别为56．0％，54．5％。56．9％，56．5％和44．9％。     

Cloning and expression of the cDNA for bovine granulocyte-macrophage colony-stimulating factor

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.     

Cloning and sequencing of a cDNA encoding the bovine FcR gamma chain

Phagocytic cells of the immune system express specific receptors for the Fc region of immunoglobulins (FcRs). In humans, most FcRs for IgG (FcgammaR), IgA (FcalphaR) and IgE (FcvarepsilonR) consist of an immunoglobulin (Ig) -binding subunit associated with a specialized signaling molecule, the FcR gamma chain. The FcR gamma chain is crucial for the transmission of intracellular signals following receptor ligation. In cattle, however, although four distinct complimentary DNAs (cDNAs) encoding IgG-binding subunits have been described (corresponding to bovine FcgammaRI, FcgammaRII, FcgammaRIII, and Fcgamma2R), virtually, nothing is known about signal transduction via bovine FcRs. Therefore, in this study, a cDNA encoding the bovine FcR gamma chain was cloned. The cDNA is 258 base pairs long and encodes a protein of 85 amino-acids. The mature protein shows high homology with the FcR gamma chains from several other species. Interestingly, the cytoplasmic domain of the bovine FcR gamma chain is one amino-acid shorter than those previously described. Cloning of a cDNA encoding, the bovine FcR gamma chain will allow for a better understanding of signal transduction processes triggered by bovine FcRs.     

中国部分地方品种鸡CD4和CD8α基因cDNA的克隆与序列分析

用Trizol分别提取4～6周龄白莱航鸡、狼山鸡、大骨鸡、北京油鸡、仙居鸡、茶花鸡、固始鸡和隐性白羽鸡胸腺细胞的总RNA，再用Oligo-dT纤维索富集mRNA后，用RT-PCR分别扩增出鸡CD4和cD8α基因cDNA。将PCR产物克隆进T载体后测序。序列分析显示不同品种鸡的CD4 cDNA序列完全一致；中国地方品种鸡与国外品种的白莱航鸡、RPL7系鸡在CD8α胞外区有6～13个氨基酸的差异，仙居鸡与其他5个中国地方品种鸡在CD8α胞外区有4～5个氨基酸的差异，狼山鸡、北京油鸡和隐性白羽鸡CD8α cDNA的核苷酸序列及推导的氨基酸序列完全一致。这些结果表明，中国地方品种鸡CD4基因序列高度保守；中国地方品种鸡与国外品种鸡的CD8α cDNA序列之间具有遗传多态性，而中国地方品种鸡之间CDSα cDNA序列遗传多态性低，为进一步研究CD4分子和CD8α链的结构和功能提供了条件。     

Cloning, sequencing, and analysis of cDNA encoding bovine granulocyte-colony stimulating factor

Neutrophils play a critical role in defending against bacterial infections. Hematopoietic growth factors are a class of regulatory cytokines that are required for stimulation, proliferation, and differentiation of blood cells. Granulocyte colony stimulating factor (G-CSF) is a cytokine that induces proliferation and maturation of precursor myeloid cells in the bone marrow into fully differentiated neutrophils. G-CSF also modulates the functional activity of mature neutrophils. Treatment with G-CSF significantly enhances neutrophil phagocytic activity and killing of bacteria and fungi. We have isolated and sequenced a cDNA clone encoding bovine G-CSF (bG-CSF) from an endothelial cell cDNA library using primers designed from ovine G-CSF. The full length cDNA is 1460 nucleotides with 585 nucleotides comprising the open reading frame. Sequence analysis shows 95% identity with ovine, 89% with porcine, 85% with human, and 76% with murine G-CSF. The deduced G-CSF protein consists of 174 amino acids with 95% identity to ovine, 86% to porcine, 81% to human, and 71% to murine. The signal peptide of G-CSF is 21 amino acids long which is nine amino acids shorter than that of human and murine G-CSF. RT-PCR analysis shows that neither freshly isolated nor ConA stimulated neutrophils express G-CSF mRNA. Mononuclear cells, however, expressed G-CSF mRNA after 48 h incubation with or without ConA stimulation.     

Molecular Cloning and Characterization of CD9 cDNA from Sheep and Cashmere Goat

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. Some CD9 cDNA have been cloned in mammals and certain fish genera in recent years, but goat and sheep counterparts of cattle, human and mouse have not been identified. To facilitate the studies, we cloned the cDNA encoding for CD9 of cashmere goat (Capra hircus) and sheep (Ovis aries), and expressed sheep CD9 in Escherichia coli cells. Structural analysis indicated for both goat and sheep that a 1123 bp cDNA spanned an open reading frame of 681 bp which predicted a protein of 226 amino acids with a typical TM4SF structure, including four highly conserved transmembrane domains, two extracellular domains and a CCG motif, which is a hallmark of the TM4SF. The predicted amino acid sequences were highly homologous to those of cattle, mouse and human CD9. Molecular phylogenetic analysis based on CD9 cDNA sequences indicated that goat and sheep CD9 were closely related to CD9 of cattle, which is in agreement with their morphological taxonomy.     

牛脂联素基因cDNA克隆

应用RT-PCR方法先提取成牛肝脏外部脂肪和犊牛大网膜前脂肪细胞培养物(第13日龄)总RNA,先后扩增出ADPN cDNA的501 bp片段基因,分别克隆到载体PMD18-T中,构建重组载体PMD18-T/牛ADPN1和PMD18-T/牛ADPN2。筛选出阳性克隆,通过限制性内切酶酶切鉴定和重组质粒PCR鉴定后对其进行测序。结果表明：ADPN1和ADPN2 2次测序结果完全一致；从脂肪组织总RNA中扩增得到501 bp片段ADPN基因,其cDNA序列与GenBank牛ADPN基因序列同源性为85%,其氨基酸同源性为96%。     

驯鹿生长素Ghrelin cDNA的克隆

从驯鹿皱胃组织中提取总RNA,根据已发表的驯鹿生长素Ghrelin基因序列设计并合成引物,通过反转录-聚合酶链式反应(RT-PCR)进行cDNA扩增,获得了300 bp的片段,重组到pBlueselect T载体,经限制性内切酶谱分析和DNA序列测定分析,确认PCR产物为Ghrelin cDNA,为进一步研究Chrelin在驯鹿体内的分布及营养因素等对其基因表达的影响奠定基础。     

牛抵抗素基因的cDNA克隆

<正>抵抗素是新近发现的一种由脂肪细胞分泌的富含半胱氨酸的多肽。人抵抗素基因定位于19p13.3,负责编码108个氨基酸残基组成的抵抗素蛋白。Tokushi确定了478bp的牛抵抗素序列,具有完整的开放阅读框架,负责编码109个氨基酸残基组成的牛抵抗素蛋白。这一序列同人的抵抗素cDNA序列具有83%的同源性。     

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。     

Cloning of canine cDNA encoding tektin

Tektins are a group of proteins that form filamentous polymers in the walls of ciliary microtubules. The cloning of canine cDNA encoding tektin, was carried out and identified from the testis of beagle dog. Canine tektin cDNA is 1,523 bp in length, has an open reading frame of 1,281 bp nucleotides encoding a protein of 426 deduced amino acids. The predicted amino acid sequence has 77% and 33-50% of homology with the murine tektin and the sea urchin tektins. The amino acid sequence RPNVELCRD and four cysteine residues were conserved in the dog, mouse and sea urchin, suggesting the functional significance of this protein domain and the amino acid residues in the tektin proteins.     

猪肌生成抑制素（MSTN）cDNA的克隆

从猪的肌生成抑制素（MSTN）编码序列中设计引物，以军牧一号猪肌细胞总PNA为模板，利用RT－PCR和嵌套PCR技术，扩增出MSTN cDNA片段。该片段全长1277bp，包含猪MSTN基因的全部编码序列。将所得片段与pMD18-T载体连接，转化到JM109大肠杆菌中，成功地筛选到阳性克隆，其质粒测序结果与文献报道的一致。经EcoR Ⅰ和Pst Ⅰ酶解分析，cDNA片段与pMD18-T载本之间既有正向插入的克隆，也有反向插入的克隆，所得到的MSTN cDNA可用于原核和真核表达载体的构建。     

鸡白细胞介素15基因的克隆及序列分析

根据GenBank中所收录的鸡白细胞介素15(ChIL 15)的cDNA序列设计特异性引物,以经ConA刺激3 h的科宝鸡脾脏淋巴细胞的总RNA 为模板,用RT PCR 方法克隆获得了ChIL 15的cDNA。ChIL 15的cD NA全长655 bp,其中第84 位~644 位是该基因的阅读框(ORF),共编码187 个氨基酸。将所克隆的序列与已报道序列相比较,二者核苷酸的同源性为100%(655/655),所编码蛋白质的氨基酸序列同源性也为100%。     

伊氏锥虫HGPRT基因cDNA的克隆及其在E.coli中的表达

根据引物设计的一般原则，参照伊氏锥虫HGPRT基因的核苷酸序列设计、合成一对引物，PCR扩增伊氏锥虫HGPRT基因cDNA。低熔点琼脂糖回收：PCR产物，并将其克隆至pGEM—T Easy载体中，经酶切、PCR鉴定和序列分析，获得重组中间质粒pGEM／HGPRT。重组中间质粒pGEM／HGPRT经Nco I和Sal I双酶切，回收目的片段HGPRT，以非融合形式插入原核表达质粒pBV220构建表达质粒pBV／HGPRT，转化大肠杆菌DH5a，42℃诱导表达，聚丙烯酰胺凝胶电泳和薄层扫描分析，表达产物HGPRT的分子量约为23kD，与理论推算值相符，表达率占总菌体蛋白的19％，经间接ELISA检测，表达产物能被伊氏锥虫阳性血清所识别。     

猪CFL2基因cDNA的克隆与序列分析

动物肌肉是人们蛋白质的主要来源之一,其基本组成单位是肌纤维.近年来,猪肉品质已成为猪遗传育种学家研究的热点和难点问题.研究表明,肌肉的组成结构和肌纤维的组织学特性与肉品品质,特别是食用品质性状密切相关.肌动蛋白是构成肌节细肌丝和细胞骨架的主要成分,并参与了生物体内一系列重要的生理活动,如肌肉收缩、细胞运动、胞质分裂等.这些过程的发生除了需要肌动蛋白以外,还需要一些与之结合的调节蛋白参与,cofilin就是其中一类低分子量的肌动蛋白结合蛋白,广泛分布于从酵母到哺乳类的生物体内.哺乳动物都表达两种cofilin基因：CFL1和CFL2,其中CFL2主要在骨骼肌和心肌表达,被认为是肌肉组织肌动蛋白装配的调节器,对正常肌肉功能和肌肉再生起着重要的作用.但目前对其研究还比较少,其在骨骼肌中的功能性作用尚未证实.本试验试图通过对猪肌肉组织CFL2基因结构及其蛋白功能的研究,进一步了解它在骨骼肌细胞生长发育中的作用,以便为后续多态性研究及今后猪的分子遗传育种提供理论基础.