[Prokaryotic expression of the major antigenic domain of equine arteritis virus GL protein and the establishment of putative indirect ELISA assay].
According to the antigenic analysis of equine arteritis virus (EAV) GL protein, one pair of primers were designed, with which the gene fragment coding the high antigenic domain of EAV GL protein was amplified from the EAV genome. The cloned gene was digested with BamH I and Xho I and then inserted into pET-32a and resulted pET-GL1. The pET-GL1 was transformed into the host cell BL21(DE3) and the expression was optimized including cultivation temperature and concentration of IPTG. The aim protein was highly expressed and the obtained recombinant protein manifested well reactiongenicity as was confirmed by Western blot. The recombinant GL1 protein was purified by the means of His * Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against EAV with the purified GL1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 9.65 microg/mL and the optimal dilution of serum was 1:80. The positive criterion of this ELISA assay is OD (the tested serum) > 0.4 and OD (the tested serum) /OD (the negative serum) > 2.0. The iGL-ELISA was evaluated versus micro-virus neutralization test. The ELISA was performed on 900 sera from which were preserved by this lab during horse entry/exit inspection, the agreement (94.1%) of these test were considered suitable for individual serological detection. In another test which 180 sera samples were detected by iGL-ELISA and INGEZIM ELISA kit respectively. The agreement ratio between the two methods is 95.6%.
Liang
Cheng-Zhu
CZ
Key Laboratory of Animal Disease Diagnosis and Immunology, Nanjing Agricultural University, Nanjing 210095, China. liangcz@163.com
Cao
Rui-Bing
RB
Wei
Jian-Chao
JC
Zhu
Lai-Hua
LH
Chen
Pu-Yan
PY
D000906:Antibodies / Q000032:analysis / Q000276:immunology; D000956:Antigens, Viral / Q000235:genetics*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007240:Equartevirus / Q000276:immunology*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011387:Prokaryotic Cells / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
D000906:Antibodies; D000956:Antigens, Viral; D014764:Viral Proteins