Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N ...Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N gene of rabies virus with the specific primers.The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection,PCR screening and gene sequencing were carried out to identify the positive clones.Finally,ExPASy and other bioinformatics softwares were used to analyze and predict the structure and biological characteristics of the N genome.Results The amplification product of RT-PCR was 1 353 bp,the recombinant plasmid pUCm-T/N was constructed,the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids （20 kinds）,the accession number submitted to the Genbank was HM756692,its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively.The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.Conclusion The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundament data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention control.展开更多
[ Objective] To find the dominant rabies virus strains that are epidemic in Guangdong Province and thus to better prevent and control rabies. [ Method] Canine brains and salivary glands collected from several regions ...[ Objective] To find the dominant rabies virus strains that are epidemic in Guangdong Province and thus to better prevent and control rabies. [ Method] Canine brains and salivary glands collected from several regions of Guangdong Province were detected by direct imrnunofluorescence assay and nested RT-PCR. The isolates were further identified by intracranial inoculation in suckling mice. The N gene was amplified by RTPCR and the sequences were aligned with those of prevalent strains in Guangxi region and vaccine strains. [ Result] Two strains were isolated from specimens collected from Guangdong Province. Their N gene sequences had a high similarity of 97.9% -99.4% to those of Guangxi strains and also had a similarity of 87.7% -88.1% to those of vaccine strains of rabies virus. [ Conclusion] Apparently healthy dogs may carry rabies virus, and the prevalence of rabies virus shows obvious regional differences.展开更多
The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared wit...The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S,sM,M and N genes open reading frame(ORF) of DX were 4 152,231,681 and 1 326 bases long respectively. There were transcription regulatory sequences(TRSs) upstream of the initiator ATG of the S,N and M genes. The amino acids sequences of S,M and N contained 30,3 and 7 potential asparagine(N) -linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06,JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.展开更多
为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。...为了建立高效、灵敏的猪流行性腹泻病毒(PEDV)检测方法,本研究从GenBank数据库中获取PEDV N基因序列,扩增出PEDV N基因标准质粒,并在N基因的保守区域内设计了一对特异性荧光定量引物,成功建立了SYBR Green I实时荧光定量PCR检测方法。经过一系列试验表明,该检测方法线性关系良好,R^(2)值为0.99;特异性强,敏感性高,最低可检测至2.23 copies/μL,比普通PCR灵敏约100倍;重复性好,组内变异系数为0.25%~0.43%,组间变异系数为0.67%~0.97%;对于各地区96份临床样品检测出PEDV阳性率为25%。本研究建立的实时荧光定量PCR检测方法为PEDV的临床诊断、流行病学调查以及定量研究提供了有效的检测工具。展开更多
基金supported by the science foundation of the Health Department of Hunan Province (Grant No A2007008 and B2009-088)the National Department Public Benefit Research Foundation (Grant No 200803014)
文摘Objective To investigate the relationship between the molecular characteristics and phylogenetic evolution of rabies N gene.Methods Saliva samples were collected from rabies cases,and RT-PCR was used to amplify the N gene of rabies virus with the specific primers.The amplifying product of RT-PCR was cloned to pUCm-T vector and transformed into E.coli XL1-Blue and then the blue-white selection,PCR screening and gene sequencing were carried out to identify the positive clones.Finally,ExPASy and other bioinformatics softwares were used to analyze and predict the structure and biological characteristics of the N genome.Results The amplification product of RT-PCR was 1 353 bp,the recombinant plasmid pUCm-T/N was constructed,the whole length of the N gene open reading frame was composed of 1 353 nucleotide residues to code 450 amino acids （20 kinds）,the accession number submitted to the Genbank was HM756692,its sequence homology of nucleotides and amino acids compared with the vaccine strain CTN-1-V was 90% and 99% respectively.The evolutionary analysis showed that the isolated strain belonged to genotype I with certain geographic regionality.Conclusion The characteristics investigation and bioinformatics analysis of Hunan0806 N gene will provide fundament data to reveal the significance of the N gene characteristics for rabies epidemiology and its prevention control.
基金supported by the grants of the Surveillance,Prevention and Control Technology Research and Demonstration Projects for Animal Rabies ( Science and Education Department of the Ministry of Agriculture,200803014) the Public Laboratory Open Fund ( Host Ecology as well as Construction of Immune Surveillance and Control System,GSKJ090103)
文摘[ Objective] To find the dominant rabies virus strains that are epidemic in Guangdong Province and thus to better prevent and control rabies. [ Method] Canine brains and salivary glands collected from several regions of Guangdong Province were detected by direct imrnunofluorescence assay and nested RT-PCR. The isolates were further identified by intracranial inoculation in suckling mice. The N gene was amplified by RTPCR and the sequences were aligned with those of prevalent strains in Guangxi region and vaccine strains. [ Result] Two strains were isolated from specimens collected from Guangdong Province. Their N gene sequences had a high similarity of 97.9% -99.4% to those of Guangxi strains and also had a similarity of 87.7% -88.1% to those of vaccine strains of rabies virus. [ Conclusion] Apparently healthy dogs may carry rabies virus, and the prevalence of rabies virus shows obvious regional differences.
基金National Basic Research Program (2004CCA00500)National High-tech Development Research Program of China (2006AA02Z440)
文摘The structure genes spike(S) ,nucleocapsid(N) ,membrane(M) ,small membrane(sM) of a porcine epidemic diarrhea virus(PEDV) strain DX isolated in Gansu province,North-west of China,were cloned,sequenced and compared with published sequences of PEDV strains. The nucleotide sequences encoding the entire S,sM,M and N genes open reading frame(ORF) of DX were 4 152,231,681 and 1 326 bases long respectively. There were transcription regulatory sequences(TRSs) upstream of the initiator ATG of the S,N and M genes. The amino acids sequences of S,M and N contained 30,3 and 7 potential asparagine(N) -linked glycosylation sites. Homologous analysis and phylogenetic trees showed that DX had the closest relationship with strains LJB/06,JS-2004-2Z and CH/HLJH/06 that were also isolated from China and indicated the prevalence of some PEDV isolates in China were widespread since the JS-2004-2Z strain originated from the south of the China,and LJB/06 and CH/HLJH/06 were isolated from northeast China. The N gene was cloned using two primers which contained Nco I and BamH I restriction enzyme sites and subcloned into expression vector pET30a. The recombinant plasmid was then transformed into E.coli Rossta. SDS-PAGE showed there was a protein of about 55kDa as expected and Western blot indicated the N protein had biological activity.
