[Expression of the VP2 gene of chicken infectious anemia virus in E. coli and analysis of immunogenicity].
The coding region of VP2 gene from Chicken Infectious Anemia was amplified from genome extracted from chicken liver tissue by PCR. PCR product was double digested with restriction enzymes BamH I and Sal I and cloned into pET-28a digested with BamH I and Sal I. Subsequently, the recombinant plasmid pET-28-VP2 was extracted and double digested with restriction enzymes BamH I and Sal I. After confirming its rightness by PCR and analysis of restriction endonucleases, the recombinant plasmid pET-28-VP2 was transformed into E. coli BL21 (DE3) strain. The culture was induced by 1 mmol/L IPTG at 37 degrees C for three hours and analyzed with SDS-PAGE. The result shows that gene encoding VP2 of CIAV was expressed successfully in E. coli and the fusion protein existed in supernatant, which was about 31kDa and showed specific immunoreactivity with anti-CIAV sera in Western blot. The fusion protein was purified by Ni2+ -affinity chromatography and quantitated by Bradford method. Then BALb/c mice were immunized with purified protein emulsified with Freund's complete adjuvant on day 0 and boosted twice on day 14 and 28 with the same dose of antigens emulsified with Freund's incomplete adjuvant, respectively. The serum isolated were examined by an enzyme-linked immunosorbant assay (ELISA) using the purified VP2 and CIAV as coating antigens and the serum could react with target protein and CIAV in ELISA detection test.
Wang
Xiao-Yan
XY
Division of Avian Infectious Diseases, National Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences , Harbin 150001, China.
Wang
Xiao-Mei
XM
Gao
Yu-Long
YL
Gao
Hong-Lei
HL
Lu
Gui-Li
GL
D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000276:immunology; D017727:Chicken anemia virus / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology
D036022:Capsid Proteins; D011994:Recombinant Proteins; C442521:VP2 protein, Chicken anemia virus