SD大鼠EGF基因cDNA序列的分子克隆

参考大鼠EGF基因序列,用PCR方法对SD大鼠EGF基因cDNA序列进行了克隆,获得了SD大鼠EGF基因的3 522bp的cDNA序列（GenBank登录号：JX149564）,其中CDS长度为3 186bp,编码了1 061个氨基酸。SD大鼠与国外报道的大鼠、小鼠、原鸡、猕猴、人等5个物种的EGF基因cDNA序列的同源性分别为97.6%、83.4%、59.7%、76.4%、73.5%,其编码蛋白的氨基酸序列同源性分别为99.2%、80.1%、52.6%、69.7%、69.4%。这一结果表明了EGF基因在进化过程中具有一定的保守性,但不同物种之间也具有特异性。通过比较SD大鼠EGF基因与GenBank数据库中发布的EGF基因的cDNA序列,本试验发现了22个SNP位点,其中9个没有改变氨基酸残基的性质,这一研究结果为EGF基因的SNPs数据库提供了新的信息。     

SD大鼠Krt71基因序列的分子克隆

参考大鼠Krt71基因序列,用PCR方法对SD大鼠Krt71基因DNA和cDNA序列进行克隆,获得SD大鼠Krt71基因的8 206bp的DNA序列(GenBank登录号:KF311105)和1 575bp的cDNA序列(GenBank登录号:KF303589),编码524个氨基酸。SD大鼠与国外报道的BN大鼠、小家鼠、家猫、家绵羊、家牛、苏门答腊猩猩和人等7个物种的Krt71基因cDNA序列的同源性分别为99.9%,95.2%,88.5%,87.5%,88.0%,88.1%,88.1%;其编码蛋白的氨基酸序列同源性分别为100.0%,98.9%,92.5%,91.5%,91.3%,91.5%,92.1%。这一结果表明Krt71基因在进化过程中具有较高的保守性,但不同物种之间也具有功能上的特异性。通过比较SD大鼠Krt71基因与GenBank数据库中发布的Krt71基因的序列,本试验发现了6个SNP位点,这些位点位于该基因的内含子序列,没有改变氨基酸的性质,这一研究结果为Krt71基因的SNPs数据库提供了新的内容。     

荣昌猪自细胞介素-6基因的克隆与序列分析

根据GenBank收录的猪白细胞介素-6（PIL-6）设计1对特异性引物，经刀豆蛋白素A（Co—nA）诱导猪淋巴细胞并提取总RNA，用1it—PCR方法扩增出荣昌猪IL-6的cDNA。将扩增基因连接到PMD18-T质粒上，经酶切鉴定和序列测定证明该序列是PIL-6。序列分析结果表明：该基因cDNA全长741bp，开放阅读框由639个核苷酸组成，推测产生的编码产物由212个氨基酸组成。核酸序列分析比对发现：荣昌猪IL-6与GenBank已发表的IL-6序列的同源性较高，为99．8％。100％，氨基酸的同源性为99．5％-100％。对荣昌猪IL-6基因氨基酸的亲水性和蛋白表面可能性进行分析，表明其与IL-6基因氨基酸序列性质一致。     

5个绵羊品种BMP6基因部分片段的多态及序列分析

根据GenBank发表的绵羊骨形态发生蛋白6（Bone morphogenetic protein6,BMP6）基因部分序列所包含的外显子5、6、7和小鼠BMP6基因外显子5、6、7序列设计3对引物,采用PCR-SSCP技术检测BMP6基因外显子5、6和7在小尾寒羊、湖羊、多赛特、特克塞尔、考力代5个绵羊品种301个个体中的单核苷酸多态性。结果发现这3对引物的扩增片段在检测的5个品种中均无多态性,说明所检测的BMP6外显子5、6、7序列比较保守。同时克隆了小尾寒羊（695bp）和济宁青山羊（699bp）部分BMP6mRNA以及小尾寒羊BMP6基因外显子3—4的内含子序列和两端部分外显子序列（785bp）。发现小尾寒羊和济宁青山羊的核苷酸和氨基酸序列差异很小,两者的核苷酸序列同源性高达99．28％,氨基酸序列同源性为98．1％,除济宁青山羊中插入一个丙氨酸外,两者只有4个氨基酸的差异。绵羊与牛、人、小鼠和大鼠的核苷酸同源性分别为97．04％、81．82％、84．68％和85．06％;氨基酸序列同源性分别为99．05％、91．43％、90．48％和92．38％,均大于90％,表明各物种BMP6核苷酸序列虽然差异较大,但氨基酸序列却非常保守。     

SD大鼠转化生长因子α基因cDNA序列的分子克隆

试验参考了SD大鼠转化生长因子α（transforming growth factor alpha, TGFα）基因序列,用PCR方法对SD大鼠TGFα基因cDNA序列进行了克隆,获得了SD大鼠TGFα基因的970 bp的cDNA序列,提交GenBank,登录号：KF366251,其中CDS长度为483 bp,共编码了160个氨基酸。SD大鼠与BN大鼠、小家鼠、黄牛、野猪、猕猴、黑猩猩、人、原鸡、鹌鹑和斑马鱼的TGFα基因编码序列的同源性分别为98.6%、95.0%、85.5%、85.3%、85.5%、86.3%、86.3%、73.2%、73.2%和56.8%,其编码蛋白的氨基酸序列同源性分别为98.8%、96.9%、90.6%、90.6%、91.2%、91.9%、91.9%、72.6%、72.6%和38.8%。这一结果表明了TGFα基因在进化过程中具有一定的保守性,但不同物种之间也具有功能上的特异性。通过比较SD大鼠与BN大鼠的TGFα基因编码序列,发现了7个SNPs位点,这一结果为TGFα基因的SNPs数据库提供了新的信息。     

马动脉炎病毒大囊膜蛋白和膜蛋白基因的克隆与序列分析

参考GenBank收录的马动脉炎病毒(EAV)读码框大囊膜蛋白基因oRF5、膜蛋白基因ORF6的核苷酸序列，分别设计了2对引物，对EAV的这两种主要结构蛋白基因进行了RT—PCR；将扩增产物克隆于pUC18通用载体，对重组质粒进行限制性内切酶分析和基因测序，证实克隆片段的可靠和准确性。测序结果，ORF5目的片段包含768bp，编码255个氨基酸组成的多肽；ORF6目的片段包含489bp，编码162个氨基酸组成的多肽。与EAVNC-002532标准毒株比较，ORF5、ORF6核苷酸的同源性分别为99．1％和99．4％；推导氨基酸的同源性分剐为96．99／6和98．29／6。     

内蒙古绒山羊 CDK2 基因的克隆与序列分析

采用同源序列克隆技术结合RT-PCR和RACE技术,首次从内蒙古绒山羊睾丸组织中克隆出羊CDK2基因的全长cDNA序列（GenBank登录号为EF035041）。结果显示：羊CDK2基因的cDNA序列长为1355bp,5′端非翻译区为174bp,3′端非翻译区为266bp,开放阅读框为894bp,编码298个氨基酸。与牛CDK2基因cDNA序列同源性为98％,氨基酸序列完全一致;和其他哺乳动物CDK2基因的cDNA序列同源性也达92％以上;氨基酸序列同源性为93％以上。说明CDK2在结构和功能上有很高的保守性。     

新城疫病毒广东分离株HN基因的克隆与序列分析

用RT-PCR方法从6个新城疫病毒(NDV)广东分离株中扩增HN基因cDNA片段。并将其克隆至pGEM-T Easy载体进行核苷酸序列测定。结果表明，6个NDV分离株HN基因片段长度均为1704bp，编码568个氨基酸；彼此间核苷酸和氨基酸同源性分别为96．0％～99．8％和98．6％～100％，与其他基因Ⅶ型毒株的氨基酸序列同源性为96．8％～98．4％；但与其他基因型毒株如D26、Ulster/67、B1、LaSota以及GB Texas的氨基酸同源性较低，为88．2％～91．0％。     

狂犬病毒Flury株糖蛋白cDNA克隆与序列分析

根据狂犬病毒糖蛋白核苷酸序列,利用Oliga软件设计两对特异性引物,通过反转录-聚合酶链式反应（RT-PCR）扩增了狂犬病毒Flury-lep糖蛋白的全长cDNA,将其插入克隆载体pMD-18T并测序。测序结果及同源性分析表明,糖蛋白cDNA长1574bp,编码524个氨基酸。狂犬病毒株Flury-lep的RGP基因序列与GenBank公布的狂犬病毒株RGP基因片段核苷酸序列的同源性为82．3％～96．3％,其编码产物的氨基酸序列同源性为88．4％～94．3％。     

鸡传染性支气管炎病毒分离株核衣壳基因克隆、序列分析和真核表达

根据GenBank公开序列自行设计一对引物，采用RT—PCR扩增出鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）W和C9001分离株完整的核衣壳（N）基因，并将其克隆至pMD18-T载体进行核苷酸序列测定和分析。结果表明，扩增的2个IBV分离株核衣壳基因片段长度均为1230bp，编码409个氨基酸，彼此间核苷酸和氨基酸同源性分别为88．0％和89．5％，与GenBank中有代表性的参考毒株相应基因核苷酸和氨基酸序列比较显示，w株核苷酸序列与GenBank中的广东分离株（AY646283）同源性最高，为94．1％，氨基酸序列同源性为94．6％；与国内部分毒株核苷酸序列同源性在86．1％～88．0％之间，氨基酸序列同源性在88．0％～90．7％之间；C9001株与国内部分毒株核苷酸序列同源性在86．4％～99．8％之间，氨基酸序列同源性在88．0％～99．8％之间。从核衣壳基因编码的氨基酸序列的系统进化树可见，W株与C9001株处于不同的进化分枝，亲缘关系较远。同时将核衣壳基因构建于真核表达质粒pVAX1中，用脂质体法将重组质粒转染入COS-7细胞中，间接免疫荧光检测出核衣壳蛋白的体外表达。研究结果为进一步研究IBV核衣壳蛋白的结构与功能以及基因工程疫苗的研制奠定了基础。     

山羊Ets-1基因cDNA的克隆与序列分析

根据GenBank已收录的牛(Bos taurus)、人(Homo sapiens)和小鼠(Mus musculus)等物种Ets-1基因序列的同源保守区域,设计特异性引物,采用RT-PCR和RACE技术,分离并克隆了西农萨能奶山羊(Capra hircus)Ets-1基因的cDNA序列。该序列全长2 263 bp(GenBank登录号HQ589338),包括5’UTR 331 bp,CDS 1 326bp和3’UTR 606 bp,编码441个氨基酸组成的蛋白质。核苷酸序列分析发现,山羊编码序列与牛、猪、人、小鼠等的相应序列同源性分别为98%、94%、92%和90%,3’UTR相应序列为96%、83%、81%和77%,5’UTR相应序列为98%、85%、82%和71%。氨基酸序列分析发现,山羊与牛、猪、人和小鼠的Ets-1的相似性较高,均在95%以上。蛋白质结构分析发现,其蛋白质分子量为50 340.8 D,等电点为5.08,具有典型的螺旋-转角-螺旋结构域,不存在跨膜结构,并且整个序列不含信号肽。     

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.     

军曹鱼Hepcidin基因全长cDNA克隆及其组织表达分析

本试验采用同源克隆和末端快速扩增(RACE)的方法,得到714 bp的军曹鱼(Rachycentron canadum) Hepcidin基因全长cDNA序列。该序列包括213 bp的5'末端非编码区(UTR)、228 bp的3'UTR及273 bp的开放阅读框(ORF),编码90个氨基酸,预测其蛋白分子质量约为10.03 ku,等电点为7.54,预测的蛋白包括信号肽、前肽和成熟肽。通过构建Hepcidin氨基酸序列的系统进化树并进行氨基酸同源性比对,结果显示军曹鱼与已知鱼类及哺乳动物Hepcidin氨基酸的同源性在24.4%~85.6%之间。实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)检测结果显示Hepcidin基因在正常军曹鱼组织中均表达,但表达量在各个组织中有所不同,其中以肝脏表达量最高。经脂多糖(LPS)和甲醛灭活的鲨鱼弧菌(Vibrio carchariae)刺激后,肝脏、头肾、脾脏中Hepcidin基因表达均上调。     

Nucleotide and deduced amino acid sequence of equine retinal and pineal gland phosducin

OBJECTIVES: To determine the full-length complementary DNA (cDNA) sequence of equine retinal and pineal gland phosducin (PHD) and to clone these sequences. SAMPLE POPULATION: Samples of equine retinal RNA. PROCEDURE: A primer set was designed for use in identifying a fragment of the equine PHD nucleotide sequence, derived from retinal RNA samples, and subsequently for use to deduce specific primers for additional examination. The full-length cDNA was determined by the method of rapid amplification of cDNA ends (RACE). For full-length cDNA, newly designed primers were used. Nucleotide sequences were analyzed by use of computer software. The deduced amino acid sequence was compared with sequences of PHD reported for other species. In addition, the sequence of equine pineal PHD was cloned. RESULTS: The cDNA nucleotide sequence for equine PHD was 1,209 base pairs (bp) in length with an open-reading frame encoding a protein of 245 amino acids and a calculated molecular mass of 28.214 kd. Similarity with amino acid sequences of PHD from other species was 89 to 93%. Sequences of equine PHD from retina and pineal gland were identical. Equine PHD contained a peptide sequence with 100% homology to an uveitopathogenic peptide reported for rat PHD. CONCLUSIONS: Equine PHD is a highly conserved protein that has homology of immunologic interest with rat PHD. These results establish a basis for studying the role of PHD in ocular inflammation of horses.     

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat (Felis domesticus) Ig epsilon constant region

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.     

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.     

猪ADAR2基因全长cDNA克隆、序列特征及表达模式分析

旨在克隆猪作用于RNA的腺苷脱氨酶2基因(ADAR2)全长cDNA序列,同时对该基因在猪不同组织中的表达规律进行探索.利用RACE(rapid-amplification of cDNA ends)对大白猪ADAR2基因mRNA全长序列进行克隆,并进行生物信息学分析;用荧光定量PCR方法检测35日龄大白猪心、肝、肺、肾...     

柳蚕异柠檬酸脱氢酶基因cDNA的克隆与表达分析

异柠檬酸脱氢酶(isocitrate dehydrogenase,IDH)是生物体内一种重要的氧化还原酶。根据已报道的烟酰胺腺嘌呤二核苷酸磷酸异柠檬酸脱氢酶(NADP-IDH)基因的保守性序列设计引物,以柳蚕(Actias seleneHubner)蛹脂肪体cDNA为模板,经PCR扩增获得了柳蚕IDH基因的部分序列。该序列长1 269 bp,编码412个氨基酸,与家蚕IDH基因的cDNA序列同源性达82.5%。柳蚕IDH与果蝇、赤拟谷盗、斑马鱼、人、大鼠、库蚊、人体虱、恶性疟原虫IDH的氨基酸序列同源性在70%左右,具有较高的保守性。半定量PCR检测结果表明,柳蚕IDH基因在蛹期不同组织中均有表达,且表达量没有显著差异。