Phylogenetic analysis of benign Theileria species based on major piroplasm surface protein (MPSP) genes from ticks of grazing cattle in Korea

Complete major piroplasm surface protein (MPSP) gene sequences of benign Theileria parasites were isolated from ticks of grazing cattle in Korea. A total of 556 tick samples were collected in five provinces: Chungbuk, Jeonbuk, Jeonnam, Gyeongbuk, and Jeju during 2010-2011. Fifteen samples from Chungbuk and Jeonnam were positive for the Theileria MPSP gene by PCR amplification using a specific primer set. A phylogenetic tree was constructed with the amplified gene sequences and 26 additional sequences published in GenBank. The benign Theileria parasites were classified into eight types, those isolated from Korean cattle ticks belonged to Types 1 (Ikeda), 2 (Chitose), 4, and 8. Types 2 and 4 were the most common types, with the rate of 40%, followed by Types 1 and 8 (with the rate of 13% and 7%, respectively). Nucleotide sequence identities of 23 theilerial MPSP sequences (15 MPSP gene sequences amplified and 8 sequences published) ranged from 67.3 to 99.8%. Multiple alignments of the deduced amino acid sequences also showed that each type was characterized by specific amino acids: 7 for Type 1, 9 for Type 2, 4 for Type 4, and 3 for Type 8.     

结缕草肌动蛋白基因全长cDNA的克隆及序列分析

根据植物肌动蛋白(Actin)基因编码区的保守序列设计引物,提取结缕草叶片的总RNA,进行RT-PCR。并采用RACE技术扩增出1560bp的Actin基因全长cDNA序列。序列分析表明,该基因的开放阅读框(ORF)为1134bp,编码377个氨基酸,5′非编码区117bp,3′非编码区309bp。所得序列与GenBank中收录的其他植物肌动蛋白核苷酸序列的一致性均在85%以上,氨基酸序列的一致性高达97%以上。将其命名为ZjACT,GenBank登录号为GU290545。根据高等植物肌动蛋白相似性构建的系统进化树显示,结缕草肌动蛋白与大麦和圆锥小麦肌动蛋白之间的亲缘关系最为密切,在进化中分化时间最为接近。进一步分离克隆了结缕草Actin基因的基因组DNA序列(登录号GU290546),它由4个外显子和3个内含子组成。本研究有助于揭示植物Actin基因家族的进化历史,为研究植物Actin基因家族功能和进化上的多样性奠定理论基础,同时也为开展草坪草和牧草Actin基因的功能分析和利用研究提供参考。     

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.     

塔乌库姆冰草肌动蛋白基因片段的克隆及序列分析

根据其他植物Actin基因的保守序列设计一对简引并性物,以塔乌库姆冰草叶片总RNA为模板,采用RT-PCR的方法扩增出Actin基因片段并连接到载体上,阳性克隆经PCR检测后进行测序。结果表明：该片段长约600bp,编码199个氨基酸,所得序列与GenBank中注册的其他植物Actin基因序列同源性均在81%以上,与其他肌动蛋白的氨基酸序列同源性达88%。     

2006—2012年鲁豫冀地区15株猪繁殖与呼吸综合征病毒的分离鉴定及ORF5/Nsp2基因的遗传变异分析

为研究鲁豫冀地区猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)的遗传变异情况,对2006—2012年来自3省区发病猪场的42份样品进行PRRSV分离鉴定,并进行了生物学特性研究和PCR鉴定,结果显示先后分离到15株PRRSV。分别采用RT-PCR扩增其ORF5基因和部分Nsp2基因并测序,与GenBank中68个ORF5序列和40个Nsp2序列的推导氨基酸序列进行比对,遗传变异分析结果表明,15株分离株均属于美洲型毒株,其中13个毒株Nsp2基因推导的氨基酸序列均存在氨基酸的不连续缺失,其ORF5基因推导的氨基酸序列与JXA1株有较高的同源性(95.5%~97.5%);SDDY2007株与疫苗株RespPRRS MLV和VR2332株亲缘关系相近,处于同一个亚群中;而HN25-2009分离株Nsp2基因推导的氨基酸序列有30个氨基酸的特征性缺失,其ORF5基因推导的氨基酸序列的遗传进化分析结果显示该分离株处于VR2332所在亚群(氨基酸同源性97.5%),具有一定特殊性。本试验结果表明,2006—2012年高致病性PRRSV是鲁豫冀地区的优势流行毒株,且存在疫苗毒株,3省区流行毒株间有一定遗传差异,但无明显地域特征。     

西伯利亚白刺肌动蛋白基因的分离与特性分析

利用兼并PCR及RACE技术克隆了西伯利亚白刺的肌动蛋白基因(Nitraria sibirica Actin, NsAct),并对其基因结构和表达特性进行了分析。所克隆的NsAct cDNA全长序列为1 831 bp,包含1 134 bp的开放阅读框,编码377个氨基酸;基因组DNA全长序列为2 913 bp,包含5个外显子和4个内含子。系统进化分析结果显示,NsAct与其他植物的肌动蛋白基因具有较高的同源性,与芒果(无患子目)亲缘关系最为接近。基因表达特性分析的结果显示,该基因在根、茎、叶中的表达量基本一致;在干旱、盐、低温及外源脱落酸胁迫下,表达量无明显变化,这些结果验证了该基因作为分子内标的可靠性。     

亚洲璃眼蜱差异表达新基因P18的克隆和鉴定

为了进行抗蜱和蜱传病疫苗的研究,本实验对亚洲璃眼蜱雌蜱吸血前后唾液腺消减文库中获取的一个全长编码基因P18进行了研究。该基因全长519bp,共编码170个氨基酸,分子量为18.36Ku,等电点为4.28。BLAST分析表明,该基因预测的氨基酸与肩突硬蜱、篦子硬蜱唾液腺的抗凝血小肽有30%～40%低度同源性。将该基因亚克隆到pET-32a( )表达载体,转化BL21(DE3)宿主菌,经IPTG诱导,重组融合蛋白以可溶性形式高效表达。将可溶性重组蛋白免疫小鼠后获得的抗血清。经免疫印迹分析表明,该重组蛋白抗体可特异性的识别半饱雌蜱唾液腺中的天然蛋白抗原,而未吸血雌蜱唾液腺中则不显现特异条带。RT-PCR结果进一步证实,该基因在蜱吸血后的唾液腺中差异表达。     

两株传染性造血器官坏死病病毒的分离及其糖蛋白基因序列分析

对所分离编号为20101008和20101032的两株传染性造血器官坏死病病毒（IHNV）的糖蛋白编码基因进行遗传进化分析,以揭示我国毒株与其他不同国家和地区毒株间的遗传进化关系。以反转录RCR（RT-PCR）法扩增其糖蛋白（G）编码基因,然后克隆入PMD-18T载体并测序。应用DNAStar和MEGA4.0软件,将20101008和20101032的G基因与多株GenBank中已发表的IHNV毒株相应基因进行比较。结果表明两株病毒间G编码基因的核苷酸和推导出来的氨基酸同源性分别为98%和81.9%,其氨基酸序列分别发生了35和20处氨基酸的替换。20101008和20101032与日本、韩国分离株同源性较高,核苷酸及推导出来的氨基酸同源性分别为93.3%～96.4%和75.9%～84.6%;两株病毒在进化关系上亲缘关系最近,属于同一分支,均为基因U型传染性造血器官坏死病病毒,但其G基因的核苷酸序列以及推导出来的氨基酸序列与已知的基因U型IHNV都有一定的差异。     

Molecular cloning and phylogenetic analysis of inflammatory cytokines of the ferret (Mustela putorius furo)

The present study determined the cDNA and deduced amino acid sequences of ferret (Mustela putorius furo) inflammatory cytokines, interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor (TNF)-alpha. The homologies of the nucleotide sequences of IFN-gamma, IL-1beta, IL-6, IL-8 and TNF-alpha of the ferret to those from other mammalian species ranged from 64.3-92.9%, 73.0-83.9, 58.1-84.8%, 58.1-89.7% and 79.0-95.0%, respectively. As distinctive amino acid residues constituting various motifs and ligand-binding sites and cysteine residues were highly conserved in ferret inflammatory cytokine proteins, ferret cytokines may have fundamentally similar functions to those of other mammals. Phylogenetic analyses based on the deduced amino acid sequences revealed that all ferret inflammatory cytokines were more closely related to those of the Carnivora order, specifically dog and cat, than to other species.     

1994—2009年我国部分地区37株鸡传染性支气管炎病毒S蛋白基因序列分析

对自1994—2009年从我国5省区免疫鸡群中分离到的37株传染性支气管炎病毒(IBV)的S蛋白基因序列进行分析,发现S1基因序列存在广泛的氨基酸替换、缺失和插入现象,大部分IBV分离株S1基因的推导氨基酸序列变异主要集中在60～63、73～74、97、128、282～299位等。S2基因较为保守,主要在裂解位点后的2～47、122～152位发生氨基酸的替换,可见IBV S基因的不断变异可能是造成本试验所调查的5省区免疫鸡群IB频发的重要原因。遗传进化分析发现本试验所调查地区近十多年来肾型毒株仍是主要流行株,没有或少有4/91型毒株流行。所调研地区鸡的腺胃炎持续广泛地发生,但是从临床腺胃病料中很少分离到IBV,可见IBV不太可能是引起腺胃炎的主要病原。     

Molecular cloning and sequence analysis of interferon-gamma and interleukin-6 from Tibetan macaque (Macaca thibetana)

The cloning and sequence analysis of Tibetan macaque IFN-(gamma) and the IL-6 cDNAs are described. The Tibetan macaque IFN-gamma and IL-6 cDNAs were found to be 498 and 639 bp in length, with open reading frames encoding 165 and 212 amino acids, respectively. Homology analyses indicated that the identity levels of nucleotide and deduced amino acid sequences of IFN-gamma among primates ranged from 93.4 to 99.2%, and 87.3 to 99.4%, respectively, and that of IL-6 ranged from 92.6 to 99.8%, and 85.4 to 99.5%, respectively. Phylogenetic analysis based on amino acid sequences showed that the Tibetan macaque is most closely related to Old World monkeys, as compared to Hominoidea and New World monkeys. These findings provide insights into the evolution of primate IFN-gamma and IL-6 and additional valuable information regarding amino acid residues essential for their biological activity.     

Comparative analysis of the putative amino acid sequences of chlamydial heat shock protein 60 and Escherichia coli GroEL

The nucleotide sequences of the gene encoding chlamydial heat shock protein 60 (cHSP60) of 7 Chlamydia psittaci strains were determined. Comparison of sequences of the cHSP60 gene among chlamydiae showed high identities of the nucleotide sequences by 81.0% or greater and of the deduced amino acid sequences by 92.2% or greater. Comparison of the amino acid sequences between chlamydia and the other bacterial HSP60s resulted in the finding of three highly conserved regions, suggesting that these regions play a role in some function. In addition, 26- or 27-functional residues in the Escherichia coli GroEL out of the 28-residues are conserved in the amino acid sequences of the cHSP60. The data suggest that the function of the cHSP60 may be the same as that of the E. coli GroEL.     

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。     

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.     

鸭Ⅰ型禽副粘病毒YH99V株HN基因的克隆和序列分析

在浙江地区进行鸭病病因的调查过程中，从患病鸭群中分离到一株引起鸭产蛋锐减而不死亡的病毒，经鉴定该病毒属于禽副粘病毒Ⅰ型，命名为YH99V株。以YH99V株的基因组RNA为模板，通过RT—PCR一步法扩增出其HN基因的cDNA片段，然后将其克隆至pMD18-T载体中，对其进行序列测定。测序后拼接出HN基因的序列长度为1785bp，该基因的ORF总长为1734bp，编码577个氨基酸。将YH99V株HN基因序列和推导的氨基酸序列与新城疫毒株的HN基因相应序列比较后发现，它们的核苷酸序列同源性分别在82．1％～99．7％，氨基酸序列同源性为87．2％～99．5％。在同源性比较的基础上，进一步绘制了Ⅰ型禽副粘病毒株HN基因的系统发育树。这对于Ⅰ型禽副粘病毒毒力基因的功能分析和该病的分子流行病调查有着重要的意义。     

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。     

10株新城疫病毒分离F基因的克隆及遗传变异分析

对10株具有一定代表性的NDV分离株的F基因进行RT-PCR扩增和序列测定,核苷酸序列及其推导的氨基酸序列比较结果表明:F基因核苷酸序列的同源性为93.6%,推导氨基酸序列同源性为95.39%;根据F基因裂解位点的氨基酸序列推测,其中2株属于弱毒株,8株属于强毒株,该结果与致病性试验测定的结果完全相符;不同年代、不同宿主分离株的F基因序列一致,高度保守.通过BLAST SEARCH比较,8株强毒株与广东鹅分离株GDGO(Y97)高度同源,处于进化树的同一分支.2株弱毒分离株与La Sota疫苗株仅有1～4个氨基酸改变,推测可能是免疫或散播La Sota疫苗株.抗原性指数分析表明HI分离株有三处明显变异,抗原位点推测分析表明H分离株比F48株和La Sota多出6个抗原位点,而Liu株抗原位点在446位后缺失.     

水貂和狐犬瘟热病毒F基因的克隆与序列分析

为探寻免疫失败的原因,对山东某养殖场免疫后的发病水貂和狐犬瘟热组织病料分别提取RNA进行RT-PCR扩增。将PCR得到的部分F基因克隆到pMD18-T载体上测序,并与疫苗株ND进行序列分析。结果表明,水貂和狐的CDV与OND的核苷酸同源性为98.6%9、8.6%,氨基酸同源性为98.0%9、8.0%。水貂和狐两者核苷酸同源性为98.0%,氨基酸同源性为95.9%。     

6株牛传染性鼻气管炎病毒BICP4基因序列的同源性分析

牛传染性鼻气管炎病毒的立即早期启动基因IER4.2编码感染细胞蛋白BICP4基因。各种疱疹病毒的BICP4基因的Ⅰ，Ⅲ，Ⅴ区变异性较大，并为其特征区。为了比较分离自不同牛种，不同部位的5种病毒株BICP4蛋白I区基因的差异性，本文根据已发表的IBBV K22毒株重复序列IRs中BICP4的基因序列设计了一特异性引物，对各毒株进行PCR扩增，均得到约540bp的片段，将其克隆，测序并连同K22株进行同源性比较。结果表明，6株病毒的核苷酸序列及氨基酸序列同源性均在98%以上，说明这些毒株重复序列中的早期基因高度保守，在一定程度上也显示出IBRV的高度保守性。     

我国近期猪瘟分子流行病学动态

利用反转录聚合酶链式反应 (RT PCR)及测序技术 ,测定 2 0 0 2年分离于我国甘肃、湖南、海南、青海等省的 9株猪瘟野毒的E2基因序列 ,并与C 株疫苗毒进行同源性比较 ,绘制CSFV系统发生树。结果表明 ,近期分离于我国的 9株野毒与C 株核苷酸序列间的同源性在80 .7%～ 99.1 %,氨基酸同源性在 89.3%～98.9%。9株野毒分属于 2个不同的组群 ,其中 5株与我国经典的石门强毒和C 株疫苗毒同属于Subgroups1 .1 ,另 4株属于与C 株有较大差异的Subgroup2 .1。