非洲猪瘟P72基因在Bac to Bac系统中的表达及抗原性鉴定  

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

作　　者：柏丽华[1] 侯绍华[1] 贾红[1] 袁维峰[1] 郭晓宇[1] 梁欠欠[1] 朱鸿飞[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物医学研究室,北京100193

出　　处：《中国畜牧兽医》2012年第3期19-23,共5页China Animal Husbandry & Veterinary Medicine

基　　金：公益性行业(农业)科研专项经费项目(200903027);中央级公益性科研院所基本科研业务费(2010js-1;2011js-3);"十一五"国家科技支撑计划重点项目(2010BAD04B02);2011年及2012年农业部动物疫情监测与防治项目

摘　　要：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1941bp,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rBac-mid-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。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.

关 键 词：非洲猪瘟 P72基因 BAC to BAC 抗原性 

分 类 号：Q78[生物学—分子生物学]