Sequencing the erbA gene of avian erythroblastosis virus reveals a new type of oncogene

Avian erythroblastosis virus (AEV) contains two distinct oncogenes, erbA and erbB . The erbB oncogene, which is homologous to a portion of the epidermal growth factor receptor, is related to the src family of oncogenes and efficiently transforms erythroblasts, whereas erbA potentiates the effects of erbB by blocking the differentiation of erythroblasts at an immature stage. This "potentiator" was sequenced; the amino acid sequence deduced from it was clearly different from the sequences of other known oncogene products and was related to carbonic anhydrases. These enzymes participate in the transport of carbon dioxide by erythrocytes, the precursors of which are main targets of avian erythroblastosis virus. A src-related oncogene such as erbB in synergy with an activated specific cell-derived gene such as erbA can profoundly affect early erythroid differentiation.     

A chimeric, ligand-binding v-erbB/EGF receptor retains transforming potential

Comparison of amino acid sequences from human epidermal growth factor (EGF) receptor and avian erythroblastosis virus erbB oncogene product suggests that v-erbB represents a truncated avian EGF receptor gene product. Although both proteins are transmembrane tyrosine kinases, the v-erbB protein lacks most of the extracellular ligand-binding domain and a 32-amino acid cytoplasmic sequence present in the human EGF receptor. To test the validity of the proposed origin of v-erbB and to investigate the functional significance of the deleted extracellular sequences, a chimeric gene encoding the extracellular and the transmembrane domain of the human EGF receptor joined to sequences coding for the cytoplasmic domain of the avian erbB oncogene product was constructed. When expressed in Rat1 fibroblasts, this reconstituted gene product (HER-erbB) was transported to the cell surface and bound EGF. Its autophosphorylation activity was stimulated by interaction with the ligand. Expression of the HER-erbB chimera led to anchorage-independent cell growth in soft agar and EGF-induced focus formation in Rat1 monolayers. Thus, it appears that v-erbB protein sequences in the chimeric receptor retain their transforming activity under the influence of the human extracellular EGF-binding domain.     

胡萝卜抗冻蛋白AFP基因的克隆与序列分析

以胡萝卜幼苗为材料,提取基因组DNA,根据GenBank中的已知序列设计引物,利用聚合酶链式反应技术(PCR)体外扩增获得胡萝卜抗冻蛋白基因(AFP),将其连接到pGEM-Teasy vector载体上,并对其进行生物学信息分析.结果表明:克隆的AFP基因片段全长1 206bp,其中编码区长1 026bp,共编码341个氨基酸,蛋白质分子量为38.048ku,等电点为5.43,该蛋白为亲水性分泌型蛋白,有信号肽存在;其蛋白质二级结构以α-螺旋、β-折叠和无规卷曲为主.与GenBank中(A91926.1)的基因序列和氨基酸序列比较,同源性分别为99.4%和97.6%,表明AFP基因克隆成功.与不同地方品种胡萝卜AFP基因做同源性比较,结果发现不同品种间的AFP基因保持着很高的内同源性.     

福氏志贺菌主动外排基因acrB的克隆与序列分析

以福氏志贺菌2a型菌株基因组为模板,经PCR扩增得acrB基因,将该基因克隆到pMD18-T Vector载体,将重组质粒进行PCR和酶切鉴定及序列测定.结果表明:测定序列与Genbank收录的福氏志贺菌参考序列同源性为99.7%,与大肠杆菌的acrB基因序列比较分析,序列同源性在98%~99%之间,说明志贺菌主动外排基因acrB在碱基序列和编码的氨基酸序列上均与大肠杆菌有很高的同源性,它可能是志贺菌引起多重耐药的主要原因.     

Direct interaction of a ligand for the erbB2 oncogene product with the EGF receptor and p185erbB2

The erbB2 oncogene encodes a 185-kilodalton transmembrane protein whose sequence is similar to the epidermal growth factor receptor (EGFR). A 30-kilodalton factor (gp30) secreted from MDA-MB-231 human breast cancer cells was shown to be a ligand for p185erbB2. An antibody to EGFR abolished the tyrosine phosphorylation induced by EGF and transforming growth factor-alpha (TGF-alpha) but only partially blocked that produced by gp30 in SK-BR-3 breast cancer cells. In two cell lines that overexpress erbB2 but do not expresss EGFR (MDA-MB-453 breast cancer cells and a Chinese hamster ovary cell line that had been transfected with erbB2), phosphorylation of p185erbB2 was induced only by gp30. The gp30 specifically inhibited the growth of cells that overexpressed p185erbB2. An antibody to EGFR had no effect on the inhibition of SK-BR-3 cell colony formation obtained with gp30. Thus, it appeared that gp30 interacted directly with the EGFR and erbB2. Direct binding of gp30 to p185erbB2 was confirmed by binding competition experiments, where gp30 was found to displace the p185erbB2 binding of a specific antibody to p185erbB2. The evidence described here suggests that gp30 is a ligand for p185erbB2.     

民猪CIRP基因的克隆与冷诱导研究

采用生物信息学结合RT-PCR的方法,克隆民猪的冷诱导RNA结合蛋白(Cold inducible RNA-binding protein,CIRP)基因,获得3个变异体序列。猪CIRP变异体1基因cDNA长1 278 bp(GenBank登录号:HQ908794),编码172个氨基酸;变异体2基因cDNA长1 653 bp(GenBank登录号:HQ908795),编码182个氨基酸;变异体3基因cDNA长1 765 bp(Genbank登录号:HQ908796),编码144个氨基酸。CIRP变异体2与变异体1相比,在其编码区的第501碱基处,出现了一段375 bp的插入片段,该插入片段的第46～48个碱基为TAG,使翻译提前终止;变异体3与变异体2相比,在其编码区的第428碱基处,出现一段115 bp插入片段,该插入片段的第5～7个碱基为TGA,使翻译提前终止。3种转录变异体的核苷酸序列同源性为86.45%,氨基酸序列同源性为87%。冷诱导后,CIRP基因在肌肉中的表达水平出现显著升高(P<0.05)。     

斑马鱼尾加压素Ⅱ受体基因组织表达的研究

尾加压素Ⅱ受体(UrotensinⅡreceptor,UT)是一种7次跨膜G蛋白偶联受体,是目前发现的具有最强收缩血管作用的尾加压素Ⅱ的特异性受体。斑马鱼中存在4种不同的UT-like基因(uts2r1,uts2r2,uts2r3和uts2r4),分别位于斑马鱼4条不同染色体上。这4种uts2r基因核苷酸序列各不相同,uts2r1位于3号染色体上,其与12号染色体上的uts2r2基因蛋白序列相似度最高为47%,与6号染色体上的uts2r3和16号染色体上的uts2r4蛋白相似度分别是41.6%和33%。通过设计特异引物检测每种uts2r基因在斑马鱼成鱼各组织中的表达情况,结果显示3号染色体上的uts2r1基因在肾脏中表达量最高,6号染色体上的uts2r2基因在脊髓中表达量最高,12号染色体上的uts2r3基因在心脏中表达量最高,而16号染色体上的uts2r4基因在背肌中表达量最高。     

禽源衣原体主要外膜蛋白基因的克隆与序列分析

根据GenBank中收录的鹦鹉热衣原体主要外膜蛋白(MOMP)基因序列设计合成1对引物,以禽源鹦鹉热衣原体基因组为模板,应用PCR扩增MOMP基因,将其克隆到pUCm-T载体上,并进行序列分析。结果显示,本株禽源鹦鹉热衣原体MOMP编码基因全长为1176 bp,与国外报道的火鸡鹦鹉热衣原体TT3株的MOMP基因同源性为91.9%,与鹦鹉鹦鹉热衣原体6BC株MOMP基因的同源性为83.1%。     

产甲酸草酸杆菌oxc基因的克隆及其蛋白结构预测

参考GenBank中已公布的产甲酸草酸杆菌(Oxalobacter formigenes,OxB)的参考序列(M77128)设计了一对扩增OXC酶蛋白基因的引物,对产甲酸草酸杆菌(ATCC 35274)的DNA抽提物进行扩增,获得了特异性的扩增片段.运用Lnternet网络上的数据库及程序,对OXC酶蛋白的理化性质、结构和功能进行生物信息学分析.结果显示克隆序列与参考序列的核苷酸序列以及推导的氨基酸序列的同源性达到99.9%和99.6%,预测该酶蛋白是分泌到胞外行使生物学功能的胞外酶,具有良好的抗原性和亲水性,二级结构是以α-螺旋为主的混合型蛋白而且其二级结构预测显示OXC酶蛋白属于硫胺素焦磷酸依赖酶家族,和其它酶家族成员一样能够发挥降解草酸的功能.     

猪巨细胞病毒的PCR检测及其gB基因特征分析

根据GenBank登录的猪巨细胞病毒(PCMV)gB基因序列设计检测引物和全长gB基因引物,对来自湖南浏阳和江西景德镇不同养殖场的猪鼻拭子样品(51份)进行PCR检测,60.78%(31/51)的鼻拭子样品扩增出了260 bp的特异性条带.再以阳性样品为模板扩增全长gB基因,并将其克隆到pMD18-T载体,核苷酸序列测定和结果分析表明,所获得PCMVgB基因序列全长2 580 bp,编码860个氨基酸,与国外PCMV分离株的核苷酸同源性为97.6% ～ 98.9%,氨基酸同源性为97.0%～98.6%,且 PCMV可分为2个群:一群为中国湖南株、英国株和德国株,命名为A群;另一群为日本株、瑞士株和西班牙株,命名为B群.抗原表位优势、亲水性、表面可及性预测分析表明,PCMVgB蛋白是理想的免疫学抗原候选蛋白.     

野猪CAPN10基因的生物信息学分析

为了研究CAPN10基因的性质和功能,使用NCBI等网站提供的一系列在线工具和软件,对其基因序列及其编码蛋白的结构和特性进行生物信息学分析。结果表明,野猪CAPN10基因CDS全长2 004 bp,编码667个氨基酸,同源性比对发现CAPN10保守性较低,对选择编码氨基酸的密码子具有偏好性;进一步的蛋白特性分析表明,野猪CAPN10是一种等电点为8.24的不稳定水溶性蛋白,无信号肽序列和跨膜结构,具有磷酸化等功能化位点及钙蛋白酶类催化基序结构。     

烟草质膜ATPase4基因的克隆、表达载体构建及表达分析

植物细胞质膜ATPase基因表达可以引起细胞向外界环境释放H⁺,从而形成细胞膜内外的电化学式梯度,促进各种离子的运输,抵抗外界的各种胁迫。为研究ATPase基因在烟草中的非生物胁迫调控机制及相关生物学功能,本研究采用同源克隆的方法,从普通烟草K326中克隆到了1个ATPase4同源性基因,成功构建ATPase4-pcambia1300过表达载体。利用生物信息学分析ATPase4基因编码蛋白的疏水性、理化性质、蛋白结构预测、信号肽预测、磷酸位点预测及同源性分析,结果显示,该基因与绒毛状烟草(Nicotiana tomentosiformis)质膜ATPase4具有99%同源性,故命名为质膜ATPase4基因。利用荧光定量PCR(qRT-PCR)测定该基因在烟草组织中的表达量及其在非生物逆境胁迫中的表达模式,结果表明,该基因在根中表达量最高,且能快速响应低钾、高盐、PEG和冷胁迫诱导,说明ATPase4基因在烟草逆境胁迫中发挥着重要的调节作用。     

萝卜抗菌肽基因AFP的克隆及其在大肠杆菌中的诱导表达

试验采用RT-PCR方法,从萝卜叶子中克隆得到抗菌肽基因AFP(antifungal protein)。该基因全长240个碱基,79个氨基酸。经测序证明克隆所得基因与GeneBank中发表的原序列一致。将此抗菌肽基因克隆到E. coli表达载体pET28a,在IPTG的诱导下表达出含有AFP的11.99 ODD,证明该AFP基因在原核表达系统中可以正常表达。     

Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene

The HER-2/neu oncogene is a member of the erbB-like oncogene family, and is related to, but distinct from, the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 189 primary human breast cancers were investigated. HER-2/neu was found to be amplified from 2- to greater than 20-fold in 30% of the tumors. Correlation of gene amplification with several disease parameters was evaluated. Amplification of the HER-2/neu gene was a significant predictor of both overall survival and time to relapse in patients with breast cancer. It retained its significance even when adjustments were made for other known prognostic factors. Moreover, HER-2/neu amplification had greater prognostic value than most currently used prognostic factors, including hormonal-receptor status, in lymph node-positive disease. These data indicate that this gene may play a role in the biologic behavior and/or pathogenesis of human breast cancer.     

长丝裂腹鱼MyoD1基因的克隆及序列分析

[目的]克隆长丝裂腹鱼的MyoD1基因,并对其进行序列分析。[方法]根据鲤鱼(Cyprinus carpio)的MyoD1基因序列设计引物,采用RT-PCR方法对长丝裂腹鱼MyoD1的全长cDNA序列进行扩增,并对该基因编码氨基酸的理化性质及同源性进行预测和分析。[结果]试验获得了包括完整ORF的长丝裂腹鱼MyoD1基因,该序列长957 bp,编码274个氨基酸,具有MRFs家族基因的典型性碱性bHLH结构;该MyoD1序列与齐口裂腹鱼、鲤鱼、斑马鱼和虹鳟等的MyoD1序列同源性较高,与人、猪、牛等哺乳动物的同源性较低。[结论]该研究为长丝裂腹鱼的肌肉发育和肉质特性形成的分子机制研究提供了基础数据,同时为青藏高原冷水鱼资源的利用与保护提供了指导意义。     

高价铁肠杆菌素受体蛋白FepA表面抗原决定簇基因的克隆及序列同源性分析

用PCR技术获取高价铁肠杆菌素受体蛋白FepA表面抗原决定簇基因,分析其在大肠杆菌种内的序列同源性。以提取的大肠杆菌基因组DNA为模板,设计一对基因特异引物,用PCR扩增的方法获得目的基因。将PCR扩增后得到的片段纯化并克隆至pMD19-T Simple Vector载体,用菌落PCR及酶切鉴定阳性菌落,对阳性菌落进行测序,并与Gene Bank公布的大肠杆菌区段进行序列同源性分析。扩增出598bp的含有受体蛋白FepA表面抗原决定簇基因的目的片段,经测序分析其种内的序列同源性为95%～99%,为今后制备针对该区段的抗体奠定了基础。     

猫血管紧张素转换酶2的三维结构模拟与真核表达

家猫能自然及人工感染SARS病毒,本研究旨在探讨家猫ACE2潜在的SARS病毒受体功能。以人血管紧张素转换酶2(ACE2)序列为模板,用同源模建法模拟了家猫ACE2的三维空间结构,发现家猫ACE2与人ACE2结构十分相似,其结合严重急性呼吸综合症(SARS)病毒囊膜纤突(S)蛋白的关键氨基酸的同源性很高(15/18)。将家猫血管紧张素转换酶2基因N端1-367 aa对应的基因片段及其跨膜区克隆到真核表达载体pcDNA3.1/myc-his( )B中,构建了表达质粒pcDNA-mA367,转染HeLa细胞,间接免疫荧光检测表明fACE2的N端片段已在细胞膜上成功表达。     

猪神经介素B和受体基因的克隆与组织分布

基于电子延伸序列,克隆并分析了猪神经介素B(NMB)和受体(NMBR)基因。猪NMBcDNA克隆片段长度为567 bp,开放阅读框架长度为366 bp,编码121个氨基酸。NMBR cDNA克隆片段长度为1 194 bp,开放阅读框架长度为1 173 bp,编码390个氨基酸。同源分析表明,猪NMB的核酸序列与牛、人、小鼠和大鼠的同源性分别为87.1%,85.2%,78.1%和76.6%,氨基酸序列的同源性分别为81%,74.4%,70.2%和70.1%;NMBR的核酸序列与牛、人、小鼠和大鼠的同源性分别为91%,89%,84.2%和83.6%,氨基酸序列的同源性分别为92.3%,90.3%,86.9%和86.4%。组织分布结果显示,猪NMB在多种组织均有分布,NMBR仅分布在大脑。克隆的猪NMB和受体基因分别注册GenBank(EU375564和EU670045)。