猪圆环病毒2型ORF3蛋白的表达及抗原性鉴定

Expression and Identification of ORF3 Protein of PCV2

为研究猪圆环病毒2型(PCV-2)ORF3蛋白的免疫活性,对其进行了表达及初步鉴定。根据NCBI上PCV-2ORF3的基因序列(FR823451)设计特异性引物,扩增后将其插入pET28a表达载体构建了重组表达质粒pET-PCV-2-ORF3,转化E.coli BL21(DE3)诱导表达后用SDS-PAGE和免疫印迹进行了鉴定。随后又用圆环病毒野毒感染猪和重组ORF2免疫小鼠的阳性血清对表达蛋白的免疫原性进行了检测。结果表明,构建成功的pET-PCV-2-ORF3表达菌被诱导后能大量表达PCV-2 ORF3蛋白。ORF3表达蛋白能与圆环病毒野毒感染猪的阳性血清发生特异性反应,与重组疫苗免疫鼠的阳性血清不能发生反应,表明ORF3蛋白具有很好的免疫原性和反应原性,有望作为圆环病毒野毒感染检测的靶抗原。

To study the function of ORF3 protein of PCV2, we expressed it and investigated its immunity activity. After desig- ning a pair of specific primers aecording to the NCBI PCV20RF3 gene sequence, it was amplified and inserted into pET28a expres- sion vector for the construction of the recombinant expression vector pET - PCV2 - ORF3. After transforming the vector into E. coli BL21 （ DE3 ）, recombinant ORF3 protein was induced to express and its immunity activity was identified with 6 this monoelonal anti- body and positive serum of wild virus infected pigs by western blot. The results showed that the successful built pET-PCV2 - ORF3 expressing bacterium was induced to express ORF3 protein. The rORF3 protein reacted with the positive serum of wild virus infected pigs in western blot, but didn＇t react with positive serum of Vaccine - inoculated mice. These suggest that ORF3 protein has good im- munogenieity and can be used as aim antigen to differentiate the inoculated pigs and infected pigs in the future.