Expression of pseudorabies virus gE epitopes in Pichia pastoris and its utilization in an indirect PRV gE-ELISA.
Pseudorabies virus glycoprotein E (PRV gE) has been recognized as a suitable diagnostic antigen for pseudorabies. In order to produce gE antigen in large quantities and at low cost, a gene fragment encoding PRV gE epitopes was expressed in Pichia pastoris expression system. SDS-PAGE and Western blotting revealed that the expression product was two recombinant proteins, approximately 38 and 32 kDa, in the culture supernatant of P. pastoris integrant 72 h after induction. Protein concentration assay showed the expression product amounted to 106.7 mg/l, accounting for 66.67% of total culture supernatant proteins. An indirect PRV gE-ELISA was then established by using the recombinant expression product as a coating antigen. Cross-reactivity assay showed that this antigen was PRV specific. Reproducibility experiment displayed good consistency. Comparison of detection results of 348 field serum samples between PRV gE-ELISA and a commercially available PRV diagnostic kit showed there was no significant difference between these two methods (P > 0.05).
Ao
Jing-qun
JQ
State Key Laboratory for Biocontrol, Sun Yat-Sen University, Guangzhou 510275, PR China. ajingqun2003@yahoo.com.cn
Wang
Jing-wen
JW
Chen
Xin-hua
XH
Wang
Xun-zhang
XZ
Long
Qing-xin
QX
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D011558:Herpesvirus 1, Suid / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011557:Pseudorabies / Q000175:diagnosis* / Q000821:virology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015203:Reproducibility of Results; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000914:Antibodies, Viral; D000956:Antigens, Viral; D000939:Epitopes; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C089580:glycoprotein E, Suid herpesvirus 1