家蚕线粒体基因组EcoRⅠ2.0kb片段的克隆及序列分析

对家蚕线粒体基因组EcoRⅠ 2 .0kb片段进行了克隆和序列分析 ,结果表明该片段包含完整的丝氨酸转移RNA基因、NADH氧化还原酶亚基Ⅰ基因、细胞色素氧化酶b亚基基因的部分序列。与果蝇 (Drosophilamelanogaster)的mtDNA进行了同源性比较 ,并根据无脊椎动物线粒体基因组密码子表 ,推定氨基酸序列。     

IBV安徽分离株膜蛋白基因的克隆及序列分析

利用设计的1对特异性引物,通过RT-PCR方法扩增出4株鸡传染性支气管炎病毒(IBV)安徽地方分离株膜蛋白M基因全长片段并进行了克隆测序。将各IBV安徽地方分离株与GenBank中注册的一些毒株M基因核苷酸序列及推导的氨基酸序列进行比较和系统进化关系分析,发现毒株间核苷酸序列同源性为88.5%~100%,其相应的氨基酸序列同源性为90.3%~100%;不同毒株间存着重组、缺失、插入及点突变等变异,从ATG至第140 bp区段的核苷酸序列变异频率最高;4株分离毒株属于同一个进化群的2个不同进化亚群,与我国常用疫苗毒株H120、M41和W 93不属同一个进化亚群。     

广西陆川猪肺炎支原体P46基因克隆及序列分析

In order to understand the sequence characteristics, basic structure and genetic variation of P46 gene which was the main immunogenic surface membrane protein gene of local prevalent Mycoplasma hyopneumoniae (Mhp) strains in Guangxi Luchuan pig.The Mhp P46 gene in positive diseased swine samples which were collected from the purebred Luchuan pig farms in Guangxi from 2011 to 2013 was amplified using PCR,and then cloned into pMD18-T vector and transformed into E.coli DH5α.We chose the positive clones and sequenced.We amplified P46 genes of four positive strains (GXLC1,GXLC2,GXLC3 and GXLC4).Use the DNAStar software to analyse the cloned sequence.The results showed that P46 gene sequence was 1104 bp coding 368 amino acids.The sequence included three Trps coded by TGA codons which weren’t termination codons and one Arg coded by CGG codons which weren’t nonsense code.The homologies of nucleotide sequences of 4 strains were 98.4% to 99.4%,and the homologies of deduced amino acid sequences were 98.6% to 99.5% with these sequences of standard J strain,232 strain,7448 strain and 168 strain.The P46 genes of these strains had highly conservation because of the high-level homologies.     

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。     

鸡传染性支气管炎病毒中国地方分离株S1基因5′端的变异

对自海南省、广西省发生鸡传染性支气管炎 (IB)鸡群分离的 4株 IBV分离株 (Ha N- 1/95、Ha N- 2 /95、GX- 1/98、GX- 2 /98)的主要免疫原纤突蛋白 S1基因经 RT- PCR扩增其 5′端约 1.2 kb的目的片段 ,将其插入载体 p MD 18- T中 ,在大肠杆菌中实现目的基因的克隆。对克隆的目的基因经限制性酶切分析及 PCR鉴定后 ,以双脱氧链终止法测定其核苷酸序列 ,并与 Gen Bank中的参考毒株 (H12 0、SD- 1/97和 Holte)相应序列作比较 ,分析其同源性。结果表明 ,Ha N- 1/95、Ha N- 2 /95、GX- 1/98及 SD- 1/97与疫苗株 H12 0的核苷酸序列同源性分别为 99.5 %、99.2 %、97.9%和99.5 % ,其推导氨基酸序列同源性分别为 99.1%、98.9%、96 .9%和 99.2 %。 GX- 2 /98与 Holte株的核苷酸序列同源率为 99.0 % ,其推导的氨基酸序列同源性为 98.6 % ,而与其他中国分离株的核苷酸序列的同源性仅为 70 %左右 ,氨基酸序列的同源性仅为 6 8%左右。     

牛支原体内蒙古分离株脂蛋白vspY基因的克隆和序列分析

旨在为进一步研究牛支原体内蒙古分离株（NM 2012）的vsp Y1、vsp Y2基因功能提供依据。根据已发表的牛支原体vsp Y1、vsp Y2基因序列设计引物,对NM 2012株的vsp Y1、vsp Y2基因进行克隆、测序,并与已发表的相关序列进行相似性比较;采用在线生物信息学分析工具对NM 2012株的vsp Y1、vsp Y2推导的氨基酸的相似性、保守结构域、跨膜结构、信号肽、脂蛋白、抗原表位进行预测。结果表明,牛支原体内蒙古分离株（NM 2012）的vsp Y1、vsp Y2基因大小分别为1 029 bp和804 bp,分别编码342和267个氨基酸组成的完整开放阅读框;vsp Y1与已发表的牛支原体vsp Y1基因相似性为99.4%~100%,其推导的氨基酸序列相似性为98.6%~99.7%,vsp Y2与已发表的牛支原体vsp Y2基因相似性为100%。根据生物信息学软件分析结果推测,vsp Y1蛋白是潜在的毒力因子。     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

伪狂犬病病毒上海株gE和gI基因的克隆及序列分析

参考Genebank发表的伪犬病病毒（Pseudorabies Virus,PRV）的gI和gE基因序列，自行设计并合成了两对引物，对PRV上海株（PRV－SH）进行PCR扩增，产物经琼脂糖电泳分析，均呈现一条约960bp和1740bp的条带，将其克隆入pGEM-T-easy载体中，进行了序旬测定，将PRV－SH株的gI基因与Rice株gI基因比较发现,核苷酸的同源性为94.7%，氨基酸的同源性为91.3%，证实为gI基因，将PRV－SH gE基因序列与Ea株、Ruce株gE基因序列进行比较，结果显示，该序列与PRV Ea株、Rice株gE基因的同源性分别为98.5%、97.5%；的氨基酸序列与Ea株，Rice株和I型单纯疱疹病毒（HSV－1）17株gE的同源性分别为97.2%、94.8%和15.6%。     

Molecular characterization of the major outer membrane protein of Haemophilus somnus

The major outer membrane protein (MOMP) of Haemophilus somnus shows antigenic and molecular mass diversity that forms the basis of a preliminary grouping system for H. somnus strains. In this study, the gene encoding MOMP of H. somnus strain 8025 was cloned in three overlapping fragments by PCR techniques, and then sequenced. The gene consists of a 1164-bp open reading frame encoding a deduced 380-amino acid protein with a 19-amino acid signal sequence, giving a mature protein with a calculated molecular mass of 39,913 Da. Significant homology was found between MOMP and porin protein sequences of bacteria in Pasteurellaceae species. When expressed in Escherichia coli, the protein from the MOMP gene directed by the T7 promoter was identical in size (approximately 40 kDa) to native MOMP and reacted with MOMP-specific antibodies. Comparisons of the MOMP gene sequences from six unrelated strains of H. somnus to that of strain 8025 revealed that the genes of three MOMP type 1 strains were highly conserved with that of strain 8025 in length and sequence. However, two MOMP type 3c strains and one MOMP type 3a strain differed markedly from the MOMP of strain 8025 in their 3'-terminal halves. Their deduced MOMP amino acid sequences differed in sequence (3c, 80.5 and 82.7% identity; 3a, 62.4% identity) and in length (3c, 384 and 376; 3a, 316), indicating that the molecular differences are the basis of antigenicity and molecular mass differences of H. somnus MOMP. In the predicted MOMP secondary structure, the variable sequences primarily mapped to putative surface-exposed loops, and a variable and surface-exposed epitope of MOMP-specific antibody was identified in the seventh-largest loop. These findings are useful for understanding the structural and immunological characteristics of H. somnus.     

传染性法氏囊病病毒超强毒HZ株和XN株VP2基因的克隆和序列比较

根据已发表的５２／７０株ＩＢＤＶ基因组序列,设计并合成了一对特异扩增ＩＢＤＶ ＶＰ２基因的引物。以陕西地区分离的ＩＢＤＶ野毒ＸＮ株,ＨＺ株为材料,以其基因组为模板利用ＲＴ－ＰＣＲ技术扩增出了１．５ｋｂ的ｃＤＮＡ产物,将ＶＰ２基因克隆于ＰＵＣ１１９质粒上,得到重组ＰＵＣ１１９质粒。     

猪瘟病毒山东惠民分离株E2基因的克隆及表达

从山东惠民某猪场病料中经RT-PCR扩增了猪瘟病毒（CSFV）E2基因,并将其克隆到pMD18-T载体。经序列测定,E2基因核苷酸序列长度为1 065bp,与GenBank中CSFV石门毒株（SHIMEN）、猪瘟兔化弱毒疫苗株（HCLV）、猪瘟ZJ7分离株E2基因核苷酸序列同源性分别为85.1%,84.2%,98.8%;氨基酸序列同源性分别为91.5%,91.5%,98.6%。将E2基因插入到大肠杆菌原核表达载体pGEX-KG,获得重组质粒pKG-E2,再转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导,受体菌能表达大小约为65 000的蛋白。Western blot显示表达的蛋白具有反应原性。     

猪繁殖与呼吸综合征病毒SCMS株MN蛋白基因的克隆与序列分析

根据GenBank中登录的美洲株ATCC VR-2332的MN蛋白基因序列,利用Oligo6.0设计一对特异性引物,以抽提的PRRSV-SCMS病毒感染细胞总RNA为模板,RT-PCR扩增出长约1.0 kb的基因片段,将其克隆入pMD 18-T载体,测序结果显示SCMS MN基因全长886 bp,包含完整的MN基因的开放阅读框,共编码297个氨基酸。PRRSV-SCMS MN基因序列与VR2332和LV株进行同源性分析结果显示,PRRSV-SCMS与VR2332和LV株之间的核苷酸序列同源性分别为99.7%和61.6%,根据M基因推导的氨基酸序列同源性分别为98.9%和78.7%,根据N基因推导的氨基酸序列同源性分别为100%和52.8%。结果表明,SCMS地方分离株与VR2332株在基因型上具有更近的亲缘关系,推测本次分离的PRRSV属于美洲型。     

cDNA of Canine Heat Shock Protein 70 (HSP70)

Full-length canine HSP70 cDNA was sequenced and the expression of HSP70 mRNA was investigated. The full-length cDNA sequence of the HSP70 gene (2322 bp) contained a single long open reading frame (1920 bp) coding a protein of 640 amino acids. The amino acid sequence of the canine HSP70 gene shared about 90-95% sequence similarity with bovine, human and mouse HSP70 proteins. Southern blot analysis with HSP70 probe gave three distinct bands of 9.4 kb, 5 kb and 4.4 kb in BamHI digests and two distinct bands of 19 kb and 4 kb in EcoRI digests. Canine HSP70 mRNA was detectable in canine peripheral blood mononuclear cells and stomach but not in liver, kidney, spleen, small intestine, large intestine and skin of dogs.     

鸭肥胖基因的分子克隆、序列分析及原核表达

根据人、小鼠、猪等动物的肥胖基因编码区序列的保守性设计1对引物，利用RT-PCR技术扩增出鸭肥胖基因的cDNA编码序列，将PCR产物插入pGEM-T载体，经：PCR和双酶切鉴定正确后进行序列测定，分析表明该cDNA序列由438个核苷酸组成，编码146个氨基酸组成的多肽，鸭与人、猪、小鼠、大鼠的核苷酸相似性分别为83％、84％、98％、94％；氨基酸的相似性分别为86％、83％、99％、96％。为了研究鸭肥胖基因体外表达的特点，构建了原核表达载体pET-28a-Ya，在大肠杆菌中进行了诱导表达。结果表明，鸭肥胖基因在大肠杆菌中进行了高效特异性融合表达，融合蛋白分子量约为20ku，其中16ku为鸭肥胖基因表达的蛋白质，经薄层扫描分析，目的蛋白约占菌体总蛋白的30％。鸭肥胖基因的克隆和表达研究，为进一步研究鸭leptin的功能与应用奠定了基础。     

牛肌肉生长抑制素基因打靶载体的构建与鉴定

本试验旨在构建用于牛肌肉生长抑制素(MSTN)基因敲除的置换型打靶载体。基于已发布的MSTN基因序列,选取第3外显子约600 bp作为靶位点,在其上、下游设计2条同源臂,分别为4.4和1.4 kb。以pPNTⅢ为骨架载体,在其2个多克隆位点处插入同源臂,构建出置换型打靶载体MSTN-KO-pPNTⅢ。结果显示,经DNA测序及酶切鉴定证实1.4 kb同源短臂和4.4 kb同源长臂均正确插入基础载体中。结果表明,成功构建出牛MSTN-KO-pPNTⅢ打靶载体。