中国生物工程杂志

The structure gene apxⅡA of ApxⅡtoxin of Actinobacillus pleuropneumoniae (APP) serotype 7 was expressed in E.coli BL-21(DE3) with prokaryotic expression vector pGEX-6p-1, the expressed product formed inclusion. The inclusion protein was washed 2-3 times with 50mmol/L Tris-HCl (PH8.0), 1mmol/L EDTA buffer contained 0.5% Triton X-100, then followed by washing one time with 1mol/L NaCl. After washing, the inclusion protein was denatuated with 6 mol/L guanidine hydrochloride. Then the solution was diluted and dialyzed against 20mmol/L Tris-HCl (pH8.3), 1mmol/L EDTA supplemented with GSH, GSSG and 0.5 mol/L L-Arginine. The solution was then concentrated by PEG 20 000 and dialyzed against 20mmol/L Tris-HCl (pH8.3), 1mmol/L EDTA. After concentration and dialysis, the rApxⅡA protein solution was purified with MicroSpin GST Purification Module kit (Amersam) according to the manufacturer's instructions. The hemolytic assay showed that the inclusion protein restored hemolytic activity after be treated by washing, denatuation, renaturation and purification, it can lysis 1% sheep erythrocyte suspension.