非洲猪瘟P72基因在Bac to Bac系统中的表达及抗原性鉴定

本研究旨在通过在Bac to Bac系统中表达非洲猪瘟病毒(ASFV)的P72基因,为进一步研究P72基因的结构、功能和免疫学特性奠定基础。本研究从含P72全长基因的菌株pGEX-6p-1-P72中扩增出P72基因全序列1941 bp,将扩增的基因连接于pFastBac HTa杆状病毒载体中,构建重组质粒pFastBac HTa-P72,转化至感受态DH10Bac中,获得重组杆粒rBacmid-P72,再将其转染至Sf9昆虫细胞,获得重组杆状病毒。经Western blotting和夹心ELISA分析,结果表明,P72在昆虫细胞Sf9中获得正确表达,重组P72蛋白可被特异性兔抗ASFV P72血清、P72单抗识别。结果提示,Bac to Bac系统表达的P72重组蛋白特异性强、活性稳定,具有良好的抗原性,为快速诊断ASF提供良好的候选抗原。

Bac to Bac Expression and Antigenicity Identification of P72 Gene of ASFV

This experiment was designed to express P72 gene of African swine fever virus(ASFV) in Bac to Bac system for further research in the structure.function and immunogenicity of the P72 gene.In this study,P72 gene complete sequence(1941 bp) was amplified by PCR using pGEX-6p-1-P72 for its template.The product was cloned into pFastBac HTa granulosis virus vector and transferred into DH10Bac competent cells.Recombined plasmid transfered into the Bacmid was named rBacmid-P72 and the rBacmid-P72 was transfected into Sf9 incect cells.The recombinant baculovirus was confirmed by PCR using M13 primers.Western blotting and sandwich ELISA confirmed that the P72 protein was expressed in Sf9 cells correctly and can be recognized by rabbit anti ASFV positive serum and P72 mAb.The results indicated that P72 protein expressed in Bac to Bac system had intensive specificity,stabile activity and good antigenicity,and can be used as candidate antigen for detecting ASF.