[Prokaryotic expression of N-terminal antigenic domain of duck plague virus gB protein and the establishment of putative indirect ELISA assay].
Based on the antigenic analysis of duck plague virus (DPV) gB protein, we designed a pair of primers to amplify the gene fragment encoding high antigenic domain of DPV N-terminal gB protein from the DPV genome. The cloned gene was digested with EcoR I and Hind III and then inserted into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level after induced with IPTG. The expressed product was analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) existed as inclusion body, which was about 42.4kDa and showed specific immunoreactivity with anti-DPV sera. The recombinant gB1 protein was purified with His-Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against DPV with the purified gB1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 6.5 microg/mL and the optimal dilution of serum was 1 : 80. The positive criterion of this ELISA assay was OD(the tested serum) > 0.4 and OD(the tested serum)/OD(the negative serum) > 2.0. The ELISA was done on 700 sera that were preserved in Shandong, Jiangsu Provinces, and were detected by igB1-ELISA and iDPV-ELISA with duck plague virus as the coating antigen respectively. The agreement ratio between the two methods was 95.6%.
Pan
Huaqi
H
Disease Diagnosis and Immunology, Nanjing Agricultural University, Ministry of Agriculture, Nanjing 210095, China. phq629@yahoo.com.cn
Cao
Ruibing
R
Liu
Lei
L
Sun
Hailiang
H
Ji
Xiangbo
X
Chen
Yongjun
Y
Chen
Puyan
P
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D003001:Cloning, Molecular; D004372:Ducks; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006564:Herpesviridae / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006566:Herpesviridae Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D008970:Molecular Weight; D011201:Poultry Diseases / Q000276:immunology / Q000821:virology*; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D014759:Viral Envelope Proteins