六株猪圆环病毒2型国内分离株的全基因组序列测定与分析   总被引：20，自引：0，他引：20  

王忠田  郭鑫  杨汉春  查振林  陈艳红 《农业生物技术学报》2005,13(1):56-60

利用PK15细胞从临床发病猪和死亡猪淋巴结中分离到6株猪圆环病毒2型(PCV2)，对其全基因组序列进行了测定和分析。结果表明，除深圳分离株(SZ)的基因组全长为1768nt外，其余5株均为1767nt；6个分离株之间全序列核苷酸同源性为98．08％；与欧、美毒株之间同源性高，介于94．4％～99．7％。  相似文献   

Study on the Change Rule of Egg Yolk Antibody in Laying Hens after First Immunization by Japanese Encephalitis Virus     

WANG Chao-qun  FANG Xu  SONG Zi-yu  HUANG Yan  PANG Jie  ZHAO Jian-guo 《中国畜牧兽医》2016,43(10):2786-2791

The purpose of this experiment was to study the immunization rule of the egg yolk antibody affected by different vaccines,immunization dose and injection ways and further to discuss the optimal immunization procedures of the laying hens for the preparation of egg yolk antibody against swine Japanese encephalitis virus.180 brown laying hens without any vaccines were selected and divided into 18 groups randomly,each group of 10 hens.Groups 1,2 were the control groups,injected with the sterile saline;Groups 3 to 10 were injected with subcutaneous or intramuscular injection,and the vaccine was injected with 0.2,0.5,1.0 and 1.5 mL successively.Groups 11 to 18 were also adopted two kinds of injection,followed by the same dose of vaccine immunization.Six eggs of each experimental group were gathered before immune day and after 3,7,10,14,18,21 and 28 days,the egg yolk antibody was extracted and the titer was determined.As a result,the egg yolk antibody titers of groups 1 to 6,11 and 12 were all 0,and no significant immune response produced;The hens from 7 to 10 groups were injected with the inactivated vaccine.After 7 days,the average antibody titer reached the peak,and the duration of the antibody was 14 days.The hens from 13 to 18 groups were injected with the attenuated virus vaccine.After 14 days,the average antibody titer reached the highest value,and the duration of the antibody was 21 days.The egg yolk antibody titers were not significantly different in the two compared experiment groups with the same injection dose but with different injection ways (P>0.05).With the same injection way of each experiment group,and the difference was significant (P>0.05).Compared with some groups with the same injection and vaccine,the titer of yolk antibody was gradually increased with the increase of the immune dose,and the difference was significant (P<0.05).The results showed that,no matter intramuscular or subcutaneous injection,in order to produce a significant immune response to hens,the immune antigen dose was 1.0 mL inactivated vaccine or 0.5 mL attenuated vaccine at least.Compared with the attenuated and inactivated vaccine,inactivated vaccine stimulated the body to produce the antibody faster,but the maintenance time was shorter;The lower dose of attenuated vaccine could stimulate the body to produce antibodies,but the speed was slower,the maintenance time was longer.  相似文献   

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody     

MAO Qian-qian  ZHOU Ling  TANG Qing-hai  BU Bin  TANG Cun-duo  JIAO Zhu-jin  YAO Lun-guang  KAN Yun-chao  YANG Jian-wei  CUI Shang-jin 《中国畜牧兽医》2016,43(7):1659-1666

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.  相似文献   

Establishment and Application of a Multiplex RT-PCR Assay for Detection of PEDV,TGEV and PRoV     

LONG Fei-xiang  SHI Kai-chuang  ZHANG Zhen  YIN Yan-wen  CHEN Han-zhong  MO Sheng-lan 《中国畜牧兽医》2016,43(10):2518-2526

In this study,a multiplex RT-PCR assay was established to differentially detect porcine epidemic diarrhea virus (PEDV),porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV) after optimization of the reaction conditions.Three pairs of primers PEDV-N,TGEV-M and PRoV-VP6 were designed for specifically amplifying PEDV N gene,TGEV M gene and PRoV VP6 gene,respectively.The assay could specifically amplify PEDV,TGEV and PRoV,but not classical swine fever virus (CSFV),porcine foot and mouth disease virus (FMDV),pseudorabies virus (PRV),porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2).The detection limits of PEDV,TGEV and PRoV standard recombinant plasmids were 1.41×10³,1.41×10² and 1.41×10³ copies/μL,respectively.The repeated reaction under the same conditions obtained uniform results.The assay was used to detect a total number of 190 clinical samples,of which 42 (22.11%) samples were positive for PEDV,58 (30.53%) samples for TGEV and 34 (17.89%) samples for PRoV,and there were mixed infection among these viruses.The results indicated that this multiplex RT-PCR assay had the advantages of sensitivity,specificity and repeatability and provided a useful tool for differential detection and epidemiological investigation of PEDV,TGEV and PRoV.  相似文献   

Establishment and Application of Nested RT-PCR Assay for Detection of Classical Swine Fever Virus     

LIU Mei-fen  WANG Shu-juan  YAN Ruo-qian  WANG Dong-fang  CAO Wei-wei  ZHAO Xue-li  LI Qin-nan  LI Ning 《中国畜牧兽医》2016,43(9):2285-2290

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Cloning and Bioinformatic Analysis of L1 Gene of BPV from Guizhou Province     

ZHANG Hai  ZHOU Bi-jun  YANG Zhong-cheng  LIAO Mei  WANG Kai-gong  WEN Ming  CHENG Zhen-tao  WANG Wei  FENG Xu-fang 《中国畜牧兽医》2016,43(11):2834-2843

In order to study and analyze L1 gene of bovine papillomavirus（BPV）in Guizhou province,the L1 gene of BPV-GZ01 strain was amplified,cloned and sequenced using bioinformatic softwares and methods,and the secondary structure,tertiary structure,B-cell preponderant epitope,conserved domains analysis, transmembrane domain and signal peptide of L1 gene were predicted.The results showed that the length of L1 gene was 1 494 bp,encoding 497 amino acids.The L1 gene of BPV-GZ01 strain shared an amino acid identities of 98.6%,99.4%,98.4%,94.4% and 91.3%,and a nucleotide identities of 99.1%,99.8%,99.4%,87.6% and 82.8% with those of BPV2,BPV2-SW01,BPV2-AKS01,BPV13 and BPV1 strains,respectively.The results of phylogenetic tree analysis indicated that there was a close relationship between BPV-GZ01 and BPV2-SW01 strains.The prediction of secondary structure of L1 protein indicated that the random coil,extended strand and alphahelix took a higher percentage.The L1 protein was supposed contain 6 potential antigen epitopes.And no transmembrane domains and no signal peptide were found.The tertiary structure of L1 protein was curved spiral structure.These results provided a theoretical basis for immunologic diagnosis and further research of nucleic acid vaccine of BPV.  相似文献   

Prokaryotic Expression and Antigenicity Analysis of Porcine Japenese Encephalitis Virus Envelope Protein Domain Ⅲ     

HOU Hua-yan  DING Ling-ling  LV Mao-jie  YANG Bao-shou 《中国畜牧兽医》2016,43(4):934-939

In order to analyze the antigenicity of porcine Japanese encephalitis virus (JEV) E protein domain Ⅲ, which was expressed by pET-28a vector with His-tag and purified through Ni-NTA, the BALB/c mice were immunized with the purified protein.We identified the antigenicity of domain Ⅲ of E protein and the anti-mice and anti-porcine JEV E Ⅲ protein specific antibody titers by SDS-PAGE, Western blotting, indirect ELISA and IFA.SDS-PAGE results showed the expressed target protein existed mainly in the form of inclusion body.Western blotting, ELISA test results showed that the protein had good reactivity with anti-serum.The mice immunized with the purified JEV E Ⅲ protein generated 1×10⁵ anti-JEV E Ⅲ protein specific antibody titers by ELISA, and the porcine immunized with the porcine JEV generated 5.1×10⁴ anti-JEV specific antibody titers.The IFA results showed that JEV E Ⅲ protein anti-serum could identify JEV antigen.The above results showed that the recombinant JEV E Ⅲ had good antigenicity.These results provided important basis for development of diagnostic antigen for JEV.  相似文献   

Isolation and Identification of BPIV3 Genotype C     

RAN Xu-hua  ZHANG Yao  CAO Si  LU Yuan-he  ZHANG Ling-ling  LIU Yang-yang  NI Hong-bo  ZHU Zhan-bo  WEN Xiao-bo 《中国畜牧兽医》2016,43(5):1368-1373

To identify the infection agents from Ningxia Hui Autonomous region, where feedlot cattle indicated bovine respiratory disease complex (BRDC), the M gene of the bovine parainfluenza virus type 3 was amplified by RT-PCR.The PCR product was ligated to pMD18-T vector and cloned to E.coli DH5α.The positive clones were sequenced and compared with the reference strains in GenBank by the molecular biology software.Sequence alignment results showed that a BPIV3 strain was isolated from the samples and named NX49, the M gene of NX49 included 1 056 nucleotides.Evolutionary analysis showed that the NX49 belonged to BPIV3 C genotype and shared 99.4% nucleotide identity with that of the SD0835 isolated in Shandong province.The characterization of the NX49 demonstrated that it was sensitive to temperature, acid and organic matter.The presence of Mg²⁺ showed no protection against the treatment at high temperature.The HA test suggested that the NX49 enables to agglutinate the guinea pig RBC at 4 ℃ and the titer was 1∶4.The study isolated a BPIV3 genotype C strain successfully, which facilitate the study of molecular evolution and epidemiology of BPIV3 in China.  相似文献   

Cloning and Sequence Analysis of HA and NA Genes of One Strain of H6N6 Subtype Avian Influenza Virus Isolated from Guizhou Province     

CHEN Jia-qi  JI Xin-qin  DUAN Zhi-qiang  WEN Ming  RUAN Yong  LEI Yun  HOU Ping 《中国畜牧兽医》2016,43(2):296-303

To investigate the epidemic situation of H6N6 subtype avian influenza virus (AIV) in Guizhou province,A/duck/Guizhou/013/2014 was isolated from Sansui duck in live poultry market of Guizhou in 2014,the hemagglutinin (HA) and neuraminidase (NA) genes of DK/GZ/14 were subjected to clone and sequence analysis.The results showed that HA gene had the highest nucleotide homologies (97.5%) with the duck-origin H6N6 subtype AIV isolated from Eastern China in 2009,and the strains of HA gene proteolytic cleavage sites was P-Q-I-E-T-R-G,which accordeol with the molecular characteristic of low pathogenic AIV (LPAIV).However,NA gene of A/duck/Guizhou/013/2014 had the highest nucleotide homologies (98.2%) with the duck-origin H6N6 subtype AIV isolated from Fujian in 2007.The phylogenetic tree showed that A/duck/Guizhou/013/2014 and Hunan strains located in the same branch,while three duck-origin H6N6 subtype AIV isolated from Guizhou in 2007 and A/duck/Guizhou/013/2014 located in the different branch for HA and NA genes in genetic evolution,which suggested that A/duck/Guizhou/013/2014 was far with the local H6N6 subtype.The results also clearly indicated that duck-origin H6N6 subtype AIV had genetic diversity in duck population in Guizhou.  相似文献   

Establishment of a Duplex Real-time RT-PCR Assay for the Differential Detection of Nipha Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus     

WANG Jian-hua  DONG Zhi-zhen  WANG Yu-ling  XIAO Yan  ZHAO Xiang-ping 《中国畜牧兽医》2016,43(2):348-355

To establish a rapid,sensitive and specific assay for the differential detection of Nipah virus (NiV) and highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV),a duplex Real-time RT-PCR was developed with specific primers and probes targeting to the special sequences of NiV M gene and HP-PRRSV nsp2 gene by optimization of reaction conditions.The performance of the assay was linear ranging from 4.6×10¹ to 4.6×10⁷ copies/μL for RNA standard control of NiV M (NiV-M-RNA) and from 4.1×10¹ to 4.1×10⁸ copies/μL for RNA standard control of HP-PRRSV nsp2 (HP-PRRSV-nsp2-RNA),and detection limits of the assay was 46 copies for the NiV-M-RNA and 4.1 copies for the HP-PRRSV-nsp2-RNA,respectively.The coefficients of variation (CVs) of both inter-assay and intra-assay repeatability were less than 2.0%,showing good repeatability.The assay was able to specifically detect NiV and HP-PRRSV simultaneously without cross-reaction with classical swine fever virus (CSFV),porcine epidemic diarrhea virus (PEDV),swine influenza virus (SIV),porcine parvovirus (PPV),pseudorabies virus (PRV) and porcine circovirus type 2 (PCV2).Of the 236 samples from pigs for both NiV and HP-PRRSV detection by the established assay,all the samples were negative for NiV,8 samples were HP-PRRSV positive.In conclusion,this assay offers a useful approach for the differential detection of NiV and HP-PRRSV in clinical specimens from the pigs.  相似文献