Optimization of the expression of hepatitis B virus e gene in Pichia pastoris and immunological characterization of the product.
Escherichia coli-derived hepatitis B e antigen (HBeAg) is widely used for serological tests of hepatitis B virus (HBV). Because it exhibits cross-reactivity with HBcAb in human sera, current antibody to HBeAg (HBeAb) immunoassays are based on competitive inhibition enzyme-linked immunosorbent assay (ELISA) rather than sandwich ELISA, which interfere with the specificity and sensitivity of HBeAb detection. Pichia pastoris has advantages of eukaryotic cells, while having the capacity of high-level secretion of foreign proteins. To explore the diagnostic suitability of recombinant HBeAg (rHBeAg), we expressed the wild type HBV e gene (wt-e-gene) and the synthetic HBV e gene (syn-e-gene; native HBV e gene modified based on synonymous codon usage bias) in P. pastoris. The recombinant antigen was secreted into the medium. The expression level of rHBeAg was enhanced by optimizing HBV e gene. The yield of syn-e-gene product was approximately five-fold greater than wt-e-gene. The protein represented 66% of the total supernatant protein, and was simply purified to 90%. P. pastoris-derived HBeAg showed high HBe antigenicity, while lacking any HBc antigenicity and cross-reactivity between all proteins derived from the culture of P. pastoris and normal human sera. P. pastoris-derived HBeAg has higher specificity and sensitivity for detection HBeAb in the diagnostic assay than the commercial HBeAb ELISA kits.
Li
Zhaoxia
Z
Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province 510630, PR China.
Hong
Guoqiang
G
Wu
Zhonghua
Z
Hu
Bo
B
Xu
Jue
J
Li
Lin
L
D024861:Genetic Enhancement / Q000379:methods*; D006513:Hepatitis B e Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*
D006513:Hepatitis B e Antigens; D011994:Recombinant Proteins