犬冠状病毒大熊猫株纤突蛋白全基因的克隆与序列分析

在国内首次对犬冠状病毒大熊猫野毒株(CCVDXMV)纤突蛋白基因进行了克隆和序列测定。该基因全长4362bp,编码1453个氨基酸,N端前18个氨基酸为推测的信号肽序列,后1435个氨基酸构成成熟蛋白。与GenBank中已发表的11个CCV毒株s基因相比,s基因核苷酸序列同源性在40．2％-99．5％之间;推导的氨基酸序列同源性在15．9％-99．O％之间。DXMV株s基因变异区主要集中在该基因前1／2处,其中350．370、439．478、1718．1818三个区域碱基变异较大,而1060．1700区却十分保守。基于s全基因及其蛋白的聚类分析表明,DXMV株与K378、NVSL和USpatent株亲源关系最近。推导的DXMV株s蛋白氨基酸序列潜在的N-联糖基化位点与CCV强毒V54相同,为34个,比Insavc．1弱毒多一个;其中第566．568位糖基化位点为多数强毒拥有而弱毒没有的。另外,DXMV株S蛋白疏水性及抗原表位与其它毒株有一定的差异,这些差异对DXMV株致病性和免疫原性等影响尚待进一步的研究。     

犬冠状病毒南京株M基因的同源重组分析

对来自腹泻犬粪样的犬冠状病毒(CCV)南京株NJ17株及参考株1-71的M基因进行了克隆、测序,并与GenBank中所有已知CCV毒株及同亚群的猪冠状病毒(TGEV)和猫冠状病毒(FCoV)代表株的M基因进行了同源性比较和系统进化分析,同时对M蛋白的结构和功能进行了预测分析.结果表明,CCV1-71与近年在中国分离到的CCV毒株V1、V2及大熊猫源的毒株具有98.9%～99.5%的同源性,说明这些毒株可能是来自同一毒株的准种.NJ17与其他中国分离株及国外分离株的同源性为87.0%～91.9%,显示国内可能存在一个相对独立进化的CCV毒株.序列比较发现,所有CCV毒株在可能的同源重组"热点"区内都有一个CTTTAG序列,与鸡传染性支气管炎病毒同源重组模板交换位点附近的特征序列相似.CCV NJ17株M蛋白在N端50氨基酸序列与FCoV 79-1683同源性高,而在后212氨基酸序列与TGEV同源性高,提示该毒株可能在M基因上曾经发生过不同病毒的同源重组.CCV M蛋白的结构及功能预测表明,所有毒株都具有分泌型信号肽,有4个螺旋跨膜区,N末端和C末端均位于膜内.M蛋白的两末端具有较强的抗原性,M蛋白上存在多种功能性氨基酸修饰位点且相对保守.N末端的氨基酸变异很大,但是功能性修饰位点相对保守,提示N末端的功能可能与构象有关.     

犬冠状病毒流行株膜蛋白基因序列分析及其表达研究

对国内分离的犬冠状病毒(CCoV)DXMV、V1和V2流行毒株膜蛋白(M)基因进行了扩增、测序和遗传进化分析.3个CCoV流行毒株M基因全长均为792bp,编码263个氨基酸,其中前17个氨基酸为信号肽.DXMV、V1和V2流行株与Insavc-1疫苗株M基因相比,核苷酸的同源性分别为92.6% 、90.9%和91.6%,推导的氨基酸序列的同源性分别为92.5%、92.0%和92.3% ,在M基因前1/3区域内存在变异,其中74-76、120-124和131-135三个区域变异较大.国内DXMV、V1、V2、NJ1和NJ1-17 5个流行株M基因核苷酸同源性为96.6%,推导的氨基酸序列同源性为96.4%,显示出很高的保守性.基于M基因的遗传进化分析表明,目前国内绝大多数CCoV流行毒株都属于CCoV基因II型,只有Fox3-1和Rac2-1两个毒株属于基因I型.另外,将DXMV株M基因亚克隆到pET28a中,在BL21(DE3)中实现了M蛋白的表达,表达量约占菌体蛋白的10.2%.     

犬冠状病毒流行株膜蛋白基因序列分析及其表达研究

对国内分离的犬冠状病毒(CCoV)DXMV、V1和V2流行毒株膜蛋白(M)基因进行了扩增、测序和遗传进化分析。3个CCoV流行毒株M基因全长均为792bp,编码263个氨基酸,其中前17个氨基酸为信号肽。DXMV、V1和V2流行株与Insavc-1疫苗株M基因相比,核苷酸的同源性分别为92.6%、90.9%和91.6%,推导的氨基酸序列的同源性分别为92.5%、92.0%和92.3%,在M基因前1/3区域内存在变异,其中74-76、120-124和131-135三个区域变异较大。国内DXMV、V1、V2、NJ1和NJ1-175个流行株M基因核苷酸同源性为96.6%,推导的氨基酸序列同源性为96.4%,显示出很高的保守性。基于M基因的遗传进化分析表明,目前国内绝大多数CCoV流行毒株都属于CCoV基因II型,只有Fox3-1和Rac2-1两个毒株属于基因I型。另外,将DXMV株M基因亚克隆到pET28a中,在BL21(DE3)中实现了M蛋白的表达,表达量约占菌体蛋白的10.2%。     

大熊猫犬瘟热病毒附着或血凝蛋白基因的序列分析

首次对犬瘟热病毒(CDV)大熊猫(GP)毒株附着或血凝蛋白(H)基因进行了序列测定并与疫苗株Onderstepoort进行了比较.我们设计合成了4对引物,对GP株进行了RT-PCR扩增与测序.H蛋白基因全长为1 946 bp,开放阅读框架(ORF)始于21-23位的ATG,终止于1 842-1 844位的TGA,编码607个氨基酸,该基因序列已被GenBank收录.将GP毒株与GenBank中疫苗弱毒株Onderstepoort进行比较,二者核苷酸序列的同源性为91.4%,推导的氨基酸序列的同源性为90.2%,GP和Onderstepoort株H蛋白的半胱氨酸残基数目均为12个且相对位置不变;疏水性有一定的变化,但推测的穿膜区位置(约35-55位氨基酸)是一致的;Onderstepoort株的H蛋白潜在的N-联糖基化位点为4个,GP株H蛋白为9个,糖基化位点的不同可能对GP株H蛋白的抗原性产生影响.     

犬瘟热病毒TN株血凝基因的克隆与序列分析

用RT PCR方法对分离的TN野毒株H基因进行了扩增 ,并将其克隆到pGEM T载体上 ,进行核苷酸序列测定。结果TN野毒株H基因ORF全长 1 ,81 5bp ,编码 60 4个氨基酸。与GenBank中己报道的 7个CDV毒株相比 ,H基因核苷酸序列的同源性在 92 %～ 99%之间 ,推导的H蛋白氨基酸序列的同源性在 91 %～ 99%之间。TN株H蛋白潜在的N 联糖基化位点为 8个 ,而Onderstepoort弱毒株H蛋白为 4个 ;其中N端第 1 9～ 2 1、 30 9～ 31 1、 5 8     

禽Ⅰ型副粘病毒f基因克隆及序列分析

用RT-PCR一步法对云南省不同禽类(鸡、鸽子)3株禽I型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽I型副粘病毒各毒株同源性为88.1%～94.9%,与疫苗株LaSota和强毒株F48E9的同源性为85.6%.所分离两株新城疫病毒在F蛋白裂解位点区(112～117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株.鸽I型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMV ZQ98-1株在这一区域的序列完全相同,揭示为中强毒株.以1 662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒属于基因Ⅶ型,鸽I型副粘病毒属于基因Ⅵ型.     

犬冠状病毒南京株M基因的同源重组分析

对来自腹泻犬粪样的犬冠状病毒(CCV)南京株NJ17株及参考株171的M基因进行了克隆、测序,并与GenBank中所有已知CCV毒株及同亚群的猪冠状病毒(TGEV)和猫冠状病毒(FCoV)代表株的M基因进行了同源性比较和系统进化分析,同时对M蛋白的结构和功能进行了预测分析。结果表明,CCV171与近年在中国分离到的CCV毒株V1、V2及大熊猫源的毒株具有98.9%～99.5%的同源性,说明这些毒株可能是来自同一毒株的准种。NJ17与其他中国分离株及国外分离株的同源性为87.0%～91.9%,显示国内可能存在一个相对独立进化的CCV毒株。序列比较发现,所有CCV毒株在可能的同源重组“热点”区内都有一个CTTTAG序列,与鸡传染性支气管炎病毒同源重组模板交换位点附近的特征序列相似。CCVNJ17株M蛋白在N端50氨基酸序列与FCoV791683同源性高,而在后212氨基酸序列与TGEV同源性高,提示该毒株可能在M基因上曾经发生过不同病毒的同源重组。CCVM蛋白的结构及功能预测表明,所有毒株都具有分泌型信号肽,有4个螺旋跨膜区,N末端和C末端均位于膜内。M蛋白的两末端具有较强的抗原性,M蛋白上存在多种功能性氨基酸修饰位点且相对保守。N末端的氨基酸变异很大,但是功能性修饰位点相对保守,提示N末端的功能可能与构象有关。     

不同致病型马立克氏病病毒DNA聚合酶基因序列比较

为了分析马立克氏病病毒(MDV)致病型与其DNA聚合酶基因的关系,本研究比较了9个不同致病型的该基因的同源性关系,这包括四种不同致病型的国际参考株即弱毒疫苗株CVI988/Ripens株、强毒株GA、超强毒株Md5和特超强毒株648A;中国疫苗株814、中国强毒参考株.Jing-1及3个中国野毒株.结果表明MDV的DNA聚合酶基因非常保守,在比较的9个毒株间,该基因上游约369个碱基的调控序列的同源性在96.7%～100%之间,该基因编码的1220个氨基酸序列的同源性在99.2%～100%之间.尽管不同毒株在一些位点上出现了氨基酸的变异,但这些变异与病毒的致病型或地域分布没有明显的关系.     

禽I型副粘病毒f基因克隆及序列分析

用RT-PCR一步法对云南省不同禽类(鸡、鸽子)3株禽I型副粘病毒F基因进行扩增和克隆,并对其f基因片段核苷酸序列进行分析,结果表明,云南省禽I型副粘病毒各毒株同源性为88.1%～94.9%,与疫苗株LaSota和强毒株F48E9的同源性为85.6%。所分离两株新城疫病毒在F蛋白裂解位点区(112～117aa)的氨基酸序列与强毒株在这一区域的序列完全相同,表明为强毒株。鸽I型副粘病毒F蛋白裂解位点区的氨基酸序列与PPMV ZQ98-1株在这一区域的序列完全相同,揭示为中强毒株。以1 662bp核苷酸绘制系统发育树,表明云南地方新城疫病毒属于基因Ⅶ型,鸽I型副粘病毒属于基因Ⅵ型。     

犬冠状病毒S糖蛋白基因重组犬2型腺病毒的构建及其免疫原性研究

首次构建了能表达犬冠状病毒纤突糖蛋白(CCVS1)的重组犬2型腺病毒(CAV-2)。用RT-PCR方法从CCVDXMV株细胞培养物中扩增出编码S糖蛋白A、B、C和D4个抗原位点的基因片段S1,将其克隆到pVAX1中,然后将含有CCVS1基因的完整表达盒(CMV-S1-PolyA)进一步定向克隆到含有CAV-2E3区的穿梭质粒pVAXE3中,构建出pVAX△E3S1。通过SalⅠ NruⅠ双酶切pVAX△E3S1回收含有目的基因的表达盒,将其克隆入含有CAV-2全基因组的骨架质粒pPoly2-CAV-2中,获得重组质粒pCAV-2-CCV-S1。ClaⅠ AscⅠ酶切pCAV-2-CCV-S1释放重组基因组,转染MDCK细胞,获得了重组病毒CAV-2-S1。该重组病毒在MDCK细胞上能产生典型的腺病毒细胞病变。通过mRNA水平和Westernblot检测,证实重组病毒能表达CCVS1蛋白。动物免疫试验表明,该重组病毒可以有效地诱导免疫犬产生抗CCV和CAV-2抗体。     

Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer,Integral Membrane and Nucleocapsid Proteins of Feline,Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU-2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.     

猪流行性腹泻病毒CH/JL毒株S基因的克隆、序列分析及线性抗原表位区的鉴定

以猪流行性腹泻病毒CH/JL毒株的RNA为模板,通过RT-PCR扩增获得的3个相互重叠的cDNA克隆覆盖了S基因,序列比对结果表明:PEDV CH/JL株S基因与CV777、Brl/87、JS、KPEDV和Chinju99毒株S基因核苷酸序列的同源性分别为96.97%、96.87%、96.41%、94.02%和93.93%,氨基酸序列的同源性分别为96.17%、95.88%、96.10%、92.36%和92.05%;分子进化树分析结果显示,PEDV CH/JL株S基因与JS毒株S基因亲缘关系最近,处于同一群。利用DNAstar Protean程序预测了PEDV CH/JL株S蛋白一个抗原表位区(83~276aa),将其克隆到原核表达载体pGEX-6p-1后转化E.coliBL21(DE3)感受态细胞,在终浓度1.0mmol/L的IPTG诱导下获得了表达,Western blot结果显示,预测的抗原表位区GST融合蛋白能与猪流行性腹泻病毒多克隆抗血清反应,提示该抗原表位区含有线性抗原表位。     

麻疹病毒F基因测序及其进化关系的初步分析

研究麻疹病毒疫苗株S191毒种及其传代的病毒溶血素F(Haemolysin,HL)基因稳定性;对该序列一些重要位点的氨基酸进行比较,推测其功能结构及生物学活性变化;同时对该基因与M蛋白基因之间的非编码序列进行比对分析。利用RT-PCR方法扩增S191减毒株MeV23、26、27代及一株流行株YunnanLC-10的F基因,测序后进行比对分析。S191传代病毒F基因序列之间核苷酸同源性为99.8%,氨基酸同源性为99.5%～99.6%;S191疫苗株与流行株之间核苷酸序列同源性达95.2%;疫苗株与流行株1003nt的非编码区序列同源性为85.0%。S191传代病毒F基因具有较高遗传稳定性,关键功能位点氨基酸未发生传代改变;1003nt非编码区序列变异速度较快。     

鸡传染性支气管炎病毒中国分离株LX4纤突蛋白基因分子特征

应用RT-PCR方法分别扩增了中国地方分离株IBV LX4 S1和S2基因并进行了基因的克隆和序列测定.结果发现,LX4 S基因由3495个核苷酸组成,编码一条1164个氨基酸残基组成的多肽.S基因编码产物裂解后形成的S1和S2亚单位分别由539和625个氨基酸残基组成.LX4 S基因推导氨基酸切割识别位点序列为HRRRR,与A2、SD/97/02和Z株相同,而与其它国内外参考毒株不同.与国内外10株已报道的具有全S基因序列的IBV参考毒株比较,LX4与国内分离毒株SD/97/02的核苷酸和氨基酸同源性最高.与32株国内外参考毒株的S1基因进化树分析比较表明,LX4与A2、SD/97/02、Z、TJ/96/02、JX/99/01和SAIBWJ等7个国内分离株在同一亚群内.在该亚群内,LX4与A2和SD/97/02亲缘关系更近,且三者在高变区和抗原表位均具有高度的同源性,而与本亚群内其它参考毒株对应的高变区和抗原表位同源性差异较大.LX4与H120 S1基因编码氨基酸的同源性虽然高于其它国外参考毒株,但同源性仍然较低,为75%.与参考毒株比较,LX4 S2基因的点突变造成其推导的氨基酸序列有11个位点发生改变,这些突变可能影响S2与S1蛋白之间的相互作用,从而影响S蛋白与特异性抗体的结合.     

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.     

Nucleotide sequence and expression of a Streptomyces griseosporeus proteinaceous alpha-amylase inhibitor (HaimII) gene.

The coding region of the alpha-amylase inhibitor (HaimII) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimII gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimII protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimII protein. The pre-HaimII protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimII protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimII gene.     

1992—1994年HIV—1国际流行株和云南株膜蛋白V3区氨基酸共有序列比较

对１９９２～１９９４年间１６个云南ＨＩＶ１株膜蛋白基因Ｖ３区进行了ＤＮＡ序列测定,经计算机ＤＮＡＳＩＳ及ＰＲＯＳＩＳ软件进行同源性分析,得出其相应的氨基酸共有序列ＹＮＶ３和两组共有序列ＹＮＶ３Ａ和ＹＮＶ３Ｂ,计算了ＹＮＶ３中每个氨基酸的保守性。分别将ＹＮＶ３Ａ和ＹＮＶ３Ｂ与世界各地的ＨＩＶ１代表株的相应序列进行了同源性比较。结果表明,ＨＩＶ１云南株膜蛋白Ｖ３区氨基酸共有序列ＹＮＶ３中每个氨基酸的平均变异度为７．６６％。两组共有序列ＹＮＶ３Ａ和ＹＮＶ３Ｂ,分别与ＨＩＶ１美欧株及泰国流行株Ｂ亚群相应序列有较高同源性。这一结果提示,在进化上云南瑞丽ＨＩＶ１流行毒株间有非常密切的关系,在这一时期该地区的流行毒株以ＨＩＶ１美欧株、泰国株Ｂ亚群及其衍生株为主。     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

单核细胞增多性李氏杆菌野毒株InlB的分子特征及其表达和纯化研究

采用PCR技术扩增单核细胞增多性李氏杆菌TA野毒株内化素B(InlB)基因,进行编码分子的序列和结构分析,并克隆入大肠杆菌表达载体pET28a中诱导表达。该基因全长1893bp,编码630个氨基酸,其中前35个氨基酸残基构成信号肽序列。在推导的InlB蛋白氨基酸序列中,从N端到C端分别包括1个α-螺旋的Cap结构域、6个富含亮氨酸的重复基序(LRR)、1个免疫球蛋白样结构域(IR)、1段B重复序列和3个串联的GW结构域,同时还存在5个潜在的N-联糖基化位点,Leu占所有氨基酸残基的10.2%。与GenBank已经报道的18个不同流行株InlB基因相比,核苷酸和推导的氨基酸序列的同源性分别在91.1%~99.6%和92.3%~99.8%之间。重组菌菌体裂解物经SDS-PAGE和Western blot分析证实该基因已经正确表达。用Ni2 亲和层析柱纯化了InlB重组蛋白。