| Porcine circovirus type2(PCV2) is the causal agent of many diseases in pigfarms collectively known as PCV2-associated disease (PCVAD). Post-weaningMultisystemic Wasting Syndrome (PMWS) is the most important PCVAD, whichcauses fever, progressive weight loss, pallor and pallor, failure, and death. PCV2wasfirst identified in western Canada in1991, and has been reported worldwidenowadays. PCV2genome is about1.7kb and contains11potential open readingframe (ORFs). ORF1and ORF2are the two major open reading frames (ORFs).ORF1encodes a replication-associated protein (Rep) which is involved in viralreplication. ORF2encodes the capsid protein (Cap) which is the major protectiveprotein. Cap protein has been related to the immune protection against PCV2.In this study, A soluble expression of recombinant Cap protein of PCV2wasobtained in E. coli BL21(DE3) after optimizing isopropyl--D-thiogalactopyranoside (IPTG) concentration, induction time and temperature.SDS-PAGE was performed to visualize protein expression, and the recombinantprotein was purified by nickel ions chromatography. The result showed that a largefraction of soluble expression of the recombinant Cap protein was obtainedfollowing induction in log phase (OD600=0.6to1.0) by a final concentration of1mmol/L IPTG under37°C for4h. Different concentrations of imidazole (20mmol/L,50mmol/L, and500mmol/L) were used in the binding, washing, and elution buffers,respectively. Western bloting showed that the recombinant Cap protein could reactspecifically with the swine poly-clonal antibody against PCV2. Non-reducedSDS-PAGE demonstrated that the recombinant Cap protein existed as both monomerof26ku and dimer of52ku.An indirect enzyme-linked immunostorbent assay (ELISA) based on therecombinant protein was developed. Using the purified recombinant protein ascoating antigen, the optimal coating condition and dilution of serum weredetermined. The results showed that the optimal concentration of recombinantprotein was2.5μg/mL. The optimal incubation condition of serum samples wasobtained with a1:400dilution at37°C for45min. And the working condition forHRP-labeled rabbit anti-protein IgG was1:1500at37°C for30min.The optimalreaction time for chromatogenic substrate was10min. A S/P ratio of X+3SD=0.416was set as a negative-positive cutoff. |
