|
|
|
| Establishment of an Indirect ELISA Based on Matrix Protein of Canine distemper virus |
|
|
|
|
Abstract Canine distemper virus (CDV), which causes fever, diarrhea, even death of Canidae and other animals, is a pathogen hazards of infectious diseases. It is important to diagnosis and control diseases using rapid and sensitive method of detecting CDV. In the work, M gene of CDV was cloned by RT-PCR from cDNA of clininal samples. The plasmid of pET-28(a)-CDV-M was constucted and identified by PCR and restricttion endonucleases digestion. The recombinant protein CDV-M were expressed in Escherichia coli by IPTG induction. The antigenicity of CDV-M were identified by Western blot. The purified recombinant protein his-CDV-M were coated as antigen, the optimal protein coating concentration was determined by ELISA square tests. The indirect ELISA method for CDV-M antibody was established and applified to detection of clinical canine serum samples. The specificity and sensitivity were completed by comparison tests of this test method and other kits. The CDV-M were expressed in E. coli BL21 (DE3). The purified protein concentration was 2.840 mg/mL. Square test showed that the optimum coated concentration of antigen was 8.88 μg/mL and the optimum dilution of antibody was 1∶160. Using this method, 223 clininal samples was detected, and the result showed that coincidence rate was 96% compared with import diagnostic kit. This study established the indirect ELISA based on M protein and can provide tools for CDV diagnosis and M protein function research.
|
|
Received: 21 July 2014
Published: 01 March 2015
|
|
Corresponding Authors:
A-Yong ZHAO
E-mail: zay503@163.com
|
|
|
|
| [1]殷震，刘景华.动物病毒学[M]. 第2版. 北京: 科学出版社，1997，743-762.
[2]SWITONSKI M，SZCZERBAL I，NOWACKA.The dog genome map and its use in mammalian comparative genomics[J][J].J Appl Genet, 2004, 45(2):195-214
[3]张永霞，王金福，张似青.犬瘟热病毒上海分离株蛋白编码基因的克隆序列与序列分析[J].中国畜牧兽医, 2008, 35(12):73-75
[4]ERIK D，DANIELLE E A，ALEXANDRE C，et al.Canine Distemper Virus Matrix Protein Influences Particle Infectivity，Particle Composition，and Envelope Distribution in Polarized Epithelial Cells and Modulates Virulence[J]. [J].J Virol, 2011, 85(14):7162-7168
[5]RELATED A，LINKSCURRAN M D，RIMA B K.The genes encoding the phosphor and matrix proteins of canine distemper virus[J].Gen Virol, 1992, 73(6):1587-1591
[6]李天松，王磊，王胜乐等.犬瘟热病毒检测方法研究进展[J].中国病原生物学杂志, 2012, 7(4):305-308
[7]NOON K F，ROGUL M，BINNL N，et al.Enzyme-linked antibody to canine distemper virus[J][J].Am J Vet Res, 1980, (41):605-609
[8]POTGIETER L N，AJIDAGBA P A.Quantitation of canine distemper virus and antibodies by enzyme-linked innunosorbent assay using protein A and monoclonal antibody capture[J][J].J Vet Diagn Inuest, 1989, 1:110-115
[9]简中友，贾赟，王全凯等.检测犬瘟热病毒抗体的重组蛋白-方法的建立及应用[J].中国预防兽医学报, 2008, 30(8):631-646
[10]MESSLING V，HARDE T C，MOENNIG V，et al.Rapid and sensitive detection of immunoglobulin M (IgM ) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based linked enzyme immunosorbent assay[J].J Clin Microbio, 1999, 37(4):1049-1056
[11]姜骞，周洁，刘家森等.犬瘟热重组蛋白抗原间接方法的建立及应用[J].中国预防兽医学报, 2008, 30(3):225-232
[12]王利利，彭建军，胡诗佳.中国宠物犬饲养存在的问题及对策[J].野生动物杂志, 2008, 29(6):317-318
[13]FUJITA K，MIURA R，YONEDA M，et al.Host range and receptor utilization of canine distemper virus analyzed viruses: Involvement of heparin-like molecular in CDV infection[J][J].Virology, 2007, 359:324-335
[14]裘云云，施朋飞，夏飞琴等.方法检测犬瘟热的流行情况[J].中国畜牧兽医, 2012, 39(6):76-78
[15]CALDERON M G，REMORINI P，PERIOLO O，et al.Detection by RT-PCR and genetic characterization of canine distemper virus from vaccinated and nonvaccinated dogs in Argentina[J].Vet Microbiol, 2007, 125(34):341-349
|
|
|
|
