Development of an indirect ELISA for serological detection of reticuloendotheliosis virus using the gp90 protein expressed in Pichia pastoris.
The present study was undertaken to express the gp90 protein of reticuloendotheliosis virus (REV) in Pichia pastoris and evaluate its potential use as a diagnostic antigen in ELISA. The full-length gp90 gene of REV was cloned into pPIC9k vector and then integrated into the chromosome of P. pastoris for induced expression. SDS-PAGE and western blot assay demonstrated that gp90 protein was expressed and secreted into the culture medium at about 100mg/L of culture under optimized condition. An indirect ELISA was then established by using the recombinant gp90 protein as the coating antigen. The optimal concentration of coated antigen was 0.1 μg/well at a serum dilution of 1:200 and the optimal positive threshold value of the assay was 0.409. Cross-reactivity assay showed that this antigen was REV specific. The reproducibility experiment displayed good consistency. Furthermore, the gp90 protein based indirect ELISA showed good correlation rates of 96.3% and 97.5% with virus neutralization test and a commercially whole virus based indirect ELISA, respectively. This study demonstrates the efficacy of recombinant gp90 protein as an antigen in ELISA for seroepidemiological study of REV infection on a large scale.
Division of Avian Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, Heilongjiang 150001, China.
Kai
K
Li
Honglei
H
Gao
Li
L
Gao
Xiaole
X
Qi
Yulong
Y
Gao
Liting
L
Qin
Yongqiang
Y
Wang
Xiaomei
X
Wang
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011201:Poultry Diseases / Q000175:diagnosis* / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results; D001356:Reticuloendotheliosis virus / Q000235:genetics / Q000276:immunology*; D012192:Retroviridae Infections / Q000662:veterinary / Q000821:virology; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins