6株猪圆环病毒2型流行毒株全基因组克隆及序列分析

参考GenBank发表的PCV-2全基因组序列,设计1对引物,通过反向PCR技术扩增出6株PCV-2流行株的全基因,并进行了序列测定分析。结果表明,6个分离株基因组全长为1 768 bp或1 767 bp,同源性比较发现,分离株之间的核苷酸同源性为95.5%~100%,与GenBank上已发表的PCV-2分离株之间的同源性为93.0%~100%,与法国分离株同源性最高。6株分离株的ORF2核苷酸及其所推导的氨基酸序列同源性分别为92.9%~100%和92.3%~100%,存在较大的变异。     

猪圆环病毒2型四川分离株ORF2基因的克隆与序列分析

对临床分离毒株PCV-2 sch2010 ORF2基因全序列进行了克隆和序列分析.结果表明,PCV-2sch2010 ORF2全基因共705 bp,编码234个氨基酸,与GenBank发表的21株PCV-2的ORF2参考序列的核苷酸氨基酸序列同源性分别为88.4%～99.7%和87.2%～99.6%;与2株PCV-1 ORF2(GU371908、FJ475129)的核苷酸序列和氨基酸序列同源性分别为58.7%～59.5%和62.8%～64.5%;PCV-2 sch2010分离株与PCV-2的欧洲分支群亲缘关系较近.     

猪圆环病毒2型江西株的全基因组克隆和序列分析

为了解江西地区猪圆环病毒2型（PCV-2）的流行和进化情况,根据GenBank上已发表的PCV-2全基因序列设计1对引物,PCR扩增后得到9条PCV-2全基因序列,并对其全基因序列核苷酸和蛋白序列进行分析,绘制遗传进化树。结果表明,江西地区流行的9株PCV-2中,基因组序列全长分为8株1 767 bp和1株1 768 bp,9株PCV-2的核苷酸同源性为94.7%~99.9%,与GenBank己发表的PCV-2分离株全基因组同源性介于94.3%~99.8%之间,而9株PCV-2的ORF1核苷酸序列同源性为96.9%~100.0%。ORF2和ORF3编码的蛋白氨基酸序列存在部分位点突变。遗传进化树显示为3种基因型：5株PCV-2b、3株PCV-2d、1株PCV-2a。本研究有助于江西地区PCV-2的监测和防制。     

猪圆环病毒2型山东株全基因组的克隆与序列分析

根据GenBank已发表的猪圆环病毒2型（PCV-2）全基因组序列,设计2对特异性引物,对山东省不同地区规模化猪场采集的疑似断奶仔猪多系统消耗综合征（PMWS）组织病料进行了PCV-2全基因组克隆和序列分析。结果表明,PCV-2核苷酸序列较稳定,不同地区8个PCV-2毒株全基因组序列均由1 767 bp组成,彼此间核苷酸序列同源性达97.3%～99.8%,亲缘关系密切;与GenBank已发表的PCV-2参考毒株的同源性介于95.6%～99.8%。而ORF2的核苷酸序列同源性只有91.6%～99.9%。     

猪圆环病毒Ⅱ型全基因组的克隆和序列分析

根据Genbank已发表的猪圆环病毒Ⅱ型(PCV-2)全基因组序列,设计两对特异性引物,建立了PCV-2全基因组的克隆和序列分析方法,并对甘肃省两个规模化养猪场采集的疑似PMWS病料进行了全基因克隆和序列分析,确定了PCV-2毒株基因序列的变异情况。结果表明,PCV-2核苷酸序列稳定,1株标准株与2株分离株全基因序列均由1 768 bp组成,彼此间序列的同源性达94.0%~99.8%,2个分离株之间同源性达94.2%~99.8%;分离株与标准株和Genbank已发表的23株PCV-2毒株参考毒株同源性达92.6%~100%。     

猪圆环病毒2型的全基因克隆和序列分析

参考GenBank中发表的猪圆环病毒2型(PCV-2)全基因序列,设计一对PCR引物,从病料中扩增获得4株PCV-2的全基因组序列,分别命名为SDNY-PCV-2、ShD-PCV-2、HBbd-PCV-2、GSLN-PCV-2,经测序确定长度均为1 767 bp。应用DNA Star序列分析表明,4个PCV-2分离株与国外部分分离株的核苷酸序列同源性达95.5%~99.8%,与PCV-1毒株的序列同源性为76.2%~77.6%。与国内外部分分离毒株序列进化树分析表明,4个PCV-2分离毒株处于不同分支中,存在一定差异。     

猪Ⅱ型圆环病毒广东分离株的全基因组克隆及序列分析

根据GenBank中猪Ⅱ型圆环病毒(PCV-2)基因序列,设计两对特异性引物.将PCV-2广东分离株(GD1株)用PK15细胞系培养,从细胞培养物中提取病毒总DNA并以之为模板,用PCR方法分段扩增病毒全基因组,分别克隆到pMD18-T载体,对阳性质粒进行序列测定.用DNAstar对序列进行拼接,得到PCV-2 GD1株基因组全序列,全长为1767个碱基,包涵2个开放阅读杠(ORF1和ORF2),分别编码与复制相关的Rep蛋白和结构蛋白Cap.通过BLAST对所测GD1株核苷核酸序列早国内外已登录GenBank中的PCV-2病毒核苷酸序更进行同源性比较,与PCV-2参考毒株的核苷酸同源性介于94.4%～99.7%之间,与德国株PCV-2(AF201897)的同源性最高,为99.7%;与猪I型圆环病毒(PCV-1)参考株(AY184287)的同源性仅为70%.     

鸭肝炎病毒浙江分离株全基因组测序及其VP1基因的序列分析

利用反转录-聚合酶链式反应(RT-PCR)技术及RACE方法扩增得到鸭肝炎病毒(DHV)浙江分离株Z10的全基因(5',3'末端序列用RACE法扩增)及4株DHV分离株的VP1基因.结果表明,分离株Z10的全基因片段长7689 bp,有1个大的开放读码框(ORF),ORF位于626～7326位核苷酸,编码2249个氨基酸.Z10分离株全基因序列与GenBank登录的6株具有代表性的DHV核苷酸序列比对,同源性94.5%～98.4%;所测得的DHV分离株的VP1基因的序列与目前GenBank上发表的具有代表性的DHv-Ⅰ VP1基因进行比对分析,结果4株Ⅰ型DHV的VP1基因cDNA长度均为714 bp,编码238个氨基酸.4株DHV-Ⅰ之间VP1基因的核苷酸序列同源性为93%～99.7%,氨基酸序列同源性为95.0%～100%;与参考毒株VP1基因的核苷酸序列同源性为92.2%～100%,氨基酸序列同源性为95.0%～100%;表明各分离毒株的亲缘关系较近,属于同一基因群.     

猪圆环病毒2型吉林分离株的全基因组序列分析

采用PCR扩增1株吉林新分离的PCV2-2017全基因组序列,经过测序拼接后进行了序列分析。毒株PCV2-2017基因全长1 767 bp,在GenBank上进行同源性比对,同源性在96%～99%之间;与法国株AF055394同源性可达99%,与美国株AF055391、四川株HM102350同源性最低,为96%。PCV2中ORF1编码Rep蛋白,核苷酸和氨基酸同源性达到99%,ORF1突变很小,相对保守;ORF2基因全长为702 bp,编码Cap蛋白,氨基酸同源性达94%～99%,核苷酸同源性与美国株比对,可达96%,与法国株和国内部分毒株同源性达99%,是变异主要发生部位,共有12处点突变;ORF3编码蛋白,核苷酸同源性高达100%,说明变异不大。基因的遗传进化分析表明,PCV2-2017吉林新分离株全基因组与法国株AF055394亲缘关系最近,同属于PCV-2b亚型;与AY322001、AB426905和国内GU370064、AY691169、AY969004、DQ180393毒株亲缘关系较远。     

陕西省部分地区猪圆环病毒2型全基因组的遗传变异分析

为了解陕西省猪圆环病毒2型(PCV2)的遗传变异情况,根据GenBank登录的PCV2全基因组序列,设计1对引物,从陕西省部分地区规模化猪场疑似PCV2感染的病猪采集病料9份,应用PCR扩增PCV2的全基因,其中6份为阳性,并对扩增的6个PCV2全基因组序列进行测序和序列分析。基因测序表明,PCV2基因组全长为1 767bp;对6株病毒序列进行同源性比较,6个毒株全基因之间核苷酸同源性为98.3%~100%,与GenBank上已发表的国内外毒株全基因组比较,同源性为96.3%~97.8%;与近几年陕西株比较发现基因变异程度不稳定,与2013年分离株(KX352154.1)同源性最高,2015年分离株(KX352159.1)次之,反而与2014年分离株(KX068219.1)最低;对6个毒株的ORF1和ORF2基因进行同源性比较,6个毒株的ORF1和ORF2基因的核苷酸同源性分别为98.1%~100%和98.2%~100%,与GenBank上已发表的国内外参考株ORF1和ORF2基因进行同源性比较,同源性分别为97.6%~99.8%和91.5~96.0%。陕西省流行的PCV2基因组较为保守,变异不大,同源性很高。     

2012年河南省猪流行性腹泻病毒分离株S1基因遗传变异分析

We designed and synthesized two pairs of specific primers amplified S1 gene by RT-PCR method to investigate the variation in S1 genes of porcine epidemic diarrhea virus (PEDV) in 2012 in Henan.S1 genes of 5 PEDV strains were cloned and sequenced, and their phylogenetic trees were analyzed from different swine breeding farms. Sequences analysis showed that S1 genes shared 98.3% to 99.5% nucleotide identities and 97.1% to 98.9% amino acids homologies among five PEDV isolates. Compared with domestic landing PEDV from 2011, the nucleotide homologies were 88.6% to 98.0% and amino acid homologies were 85.3% to 98.7%. Compared with CV777, the nucleotide homologies were 88.7% to 89.1% and amino acid homologies were 87.3% to 88.4%. Homology analysis showed that S1 genes of 5 isolates shared the same genetic mutation, there were the same insertions and deletions. Compared with domestic mutant strains landed from 2011, there was no tendency. However, compared with CV777, there were three nucleotide insertions from 163 to 166 bp, nine nucleotide insertions from 173 to 174 bp, three nucleotide insertions from 405 to 406 bp and three nucleotide deletions from 463 to 464 bp. These insertions and deletions of nucleotides led to its corresponding changes in encoding amino acids. Phylogenetic analysis showed that S1 genes of 5 PEDV strains belonged to the third group. However compared with part of PEDV first group domestic mutant strains landed in 2011, the nucleotide homologies were 88.6% to 89.3% and amino acid homologies were 85.3% to 86.9%. CV777 was the second group. The results showed that S1 genes of PEDV prevalent strain exist in first and third groups, but mainly prevailing third group strains, the pathogenic and antigenic differences of these strains should be further studied.     

猪链球菌9型cps9G基因的克隆与序列分析

根据GenBank中猪链球菌9型(SS9)基因的核酸序列,设计一对引物,采用PCR方法从确诊为猪链球菌的阳性样品中扩增cps9G基因片段,将其克隆到pMD18-T载体上,转化DH5a感受态细胞,提取重组质粒pMD18T-cps9G,经PCR和酶切鉴定后测序,并与GenBank上SS9相应序列进行同源性分析.结果表明,经PCR扩增,鉴定为SS9的有8株.序列分析发现,8株SS9的cpsgG片段的核苷酸序列较稳定,该基因片段长度均为562 bp,彼此间的核苷酸同源性达96.8%～99.8%,亲缘关系密切;与GenBank上已发表的SS9参考毒株的同源性介于96.0%～98.6%.     

Cloning and Sequence Analysis of Meq Gene of Marek's Disease Virus Guangxi Strain

To investigate genetic variation of Marek's disease virus(MDV) in Guangxi province, three isolates of MDV were isolated from infected chicken.One pair of primers for amplifying Meq gene of MDV was designed according to nucleotide sequence in GenBank, Meq gene of the isolates were amplified by PCR, and then cloned, sequenced and compared with reference MDV strains published in GenBank.The results showed that Meq gene from all of the MDV isolates consisted of 1020 bp, coding for 339 amino acids.Compared with reference strains published in GenBank, the sequences of Meq gene in different isolates were relatively conserved and the homologies of nucleotide and amino acid sequence of the isolates were 83.8% to 99.9% and 88.4% to 99.6%, respectively.The proline-rich repeats of Meq gene of the MDV isolates had site mutations, and it was related to MDV's virulence.The isolate were nearly related to YL and GXY2, and far away from RB1B, GA, Md5, 648A and the immune strain phylogenetically.The study would provide research materials for the prevalence, genetic variation, protection and control of MDV in China.     

鸡马立克氏病病毒广西株Meq基因的克隆与序列分析

为研究鸡马立克氏病病毒(MDV)广西流行株的遗传变异情况,本研究从广西发病鸡中分离鉴定了3株MDV。参照GenBank中MDV的核苷酸序列设计1对引物,利用PCR技术对分离毒株的Meq基因进行了克隆、序列测定,并与GenBank中发表的国内外参考毒株进行比对分析。结果显示, Meq基因序列全长为1020 bp,编码一条由339个氨基酸组成的多肽,分离株与国内外MDV参考毒株相比,不同MDV株的Meq基因序列相对较保守,它们之间核苷酸同源性为83.8%~99.9%,氨基酸同源性为88.4%~99.6%。3株MDV分离株Meq基因在相关报道中提到的与毒力相关的脯氨酸重复区存在点突变。3株分离株与国内参考株YL、GXY2关系较近,与参考株RB1B、GA、Md5、648A及疫苗株亲缘关系较远。该研究为中国MDV的流行、遗传变异及防控研究提供了材料。     

11株新城疫病毒广西分离株NP基因的克隆和序列分析

根据基因库中新城疫病毒(NDV)的NP基因序列设计了1对特异性引物,应用RT-PCR技术对广西在2000～2003年暴发新城疫的鸡群中分离的11株NDV毒株NP基因进行RT-PCR扩增和序列测定,拼接出11个NDV广西分离株的NP基因的全序列,10个NDV广西分离株的NP基因阅读框的核苷酸序列全长均为1470 bp,编码489个氨基酸,它们的NP基因核苷酸全序列及推导的氨基酸全序列与10个已发表的NDV参考株的NP基因全序列比较分析结果表明:核苷酸序列同源性为84.8%～98.2%,氨基酸同源性为89.8%～99.4%.     

致犊牛肺炎牛支原体南疆株的分离鉴定及oppF基因序列分析

本研究旨在调查新疆喀什某规模化奶牛场的犊牛死亡原因,并确定病原体.无菌采集3份因肺炎死亡的犊牛肺脏病料样品.采用牛支原体专用液体培养基和1.0%牛支原体琼脂固体筛选培养基从3份病死犊牛肺脏病料中分离得到2株牛支原体(Mycoplasma bovis,M.bovis),分别命名为M.bovis-NJ-1和M.bovis-NJ-2.通过菌落形态学观察、特异性PCR和oppF测序比对对分离株进行鉴定.结果显示,2个分离株在固体培养基上的菌落呈现典型的"煎蛋状",且Dienes染色特点符合牛支原体菌落着色特征,中心呈深蓝色;PCR能扩增出牛支原体特异的448 bp目的片段;2个分离株的oppF基因序列与牛支原体国际标准株PG45的同源性分别为96.7%和95.3%.结果表明,引起犊牛发病死亡的病原是牛支原体,本研究为犊牛支原体肺炎的快速诊断和防制提供依据.     

柑橘裂皮类病毒江西分离物（CEVd-RJ）全长cDNA序列分析

运用RT-PCR技术对柑橘裂皮类病毒江西分离物CEVd-RJ的全长cDNA序列进行扩增、克隆，并对其片段进行序列测定。将获得的371bp的全长cDNA序列与GenBank登录的柑橘裂皮类病毒相应序列进行比对分析，结果发现江西分离物CEVd-RJ与国内柑橘裂皮类病毒湖北分离物CEVd-HB的同源性为99.7%，与广东分离物CEVd-YC的同源性为90.8%，同国外9个分离物间的同源性在89.0%~98.6%之间。序列差异分析发现柑橘裂皮类病毒江西分离物CEVd-RJ的全长cDNA序列比广东分离物CEVd-YC少一个碱基，有36个位点碱基发生了变化，与湖北分离物CEVd-HB存在一个碱基位点的差异。     

NDV 江苏分离株F基因的克隆与序列分析

为比较NDV江苏分离株的遗传变异特征,应用RT-PCR扩增出新城疫病毒（NDV）江苏徐州株（JSXZ）、江苏宿州株（JSSZ）、江苏连云港株（JSLYG）和江苏淮安株（JSHA）的融合蛋白F基因的全长核苷酸,将其分别克隆至pMD18-T载体中。将获得的阳性重组质粒进行序列测定后,推导出其氨基酸序列,进行分析和比较。结果表明,所获得的4个分离株F基因完整的开放阅读框长度为1662bp,共编码F蛋白的553个氨基酸;4个毒株与常用疫苗株之间核苷酸序列同源性只有69．3％～80．8％。JSXZ株和JSSZ株F基因的裂解位点为112-RRQK／RRF-117,符合强毒株裂解区氨基酸组成的特征。以lbp～374bp的核苷酸序列绘制系统发育树分析表明,JSXZ和JSSZ株NDV为基因Ⅷ型,JSLYG和JSHA株NDV为基因Ⅸ型。     

Cloning and Sequence Analysis of N and M Genes of Avian Infectious Bronchitis Virus Guangxi Strain

To investigate genetic variation of avian infectious bronchitis virus (IBV) in Guangxi province,one strain of IBV was isolated from chicken.Two pairs of primers for amplifying the N and M genes of IBV were designed according to the sequences in GenBank.The N and M genes of the strain were amplified by RT-PCR,and they were proved to be the N and M genes of IBV by cloning,sequencing and compared with reference IBV strains published in GenBank.The results showed that the N gene from the IBV isolate consisted of 1 230 bp,coding 409 amino acids.The M gene from the IBV isolate consisted of 678 bp,coding 225 amino acids.The sequence analysis of N gene showed that it shared 87.2% to 93.3% nucleotide homologies and 90.0% to 94.4% deduced amino acid sequence homologies with IBV strains from GenBank.The M gene sequence analysis showed that it shared 83.6% to 91.0% nucleotide homologies and 82.7% to 92.9% deduced amino acid sequence homologies.The phylogenetic tree analysis showed that it was closely related to BJ and LX4 strains,and were clustered into one group;But with the distant relatives from other strains of IBV.These results suggested that the isolate was a new variant of IBV.