Abstract：In order to preparate polyclonal antibodies of ORF3 and ORF4 of Tobacco bushy top virus (TBTV) , ORF3 and ORF4 fragments were cloned from TBTV-BSLLi isolate by RT-PCR, and sub-cloned into pMD18-T. After correct sequencing, the ORF3 and ORF4 fragments were inserted into prokaryotic expression vector pET28a(+). Then the recombinant vector was transformed into Esherichia coli BL21(plysS) through heat shock, and 6×His-tagged ORF3 and ORF4 expression were induced by IPTG. The high purity protein of ORF3 and ORF4 were obtained through Ni affinity chromatography column. Their high titer specific antiserum were obtained by immunizing rabbit using the purity proteins．The DAS-ELISA results indicated that the antiserum could be used for specific detection of TBTV from field samples of tobacco(Nicotiana tabacum L.) and transmission vector. It provides the basic condition for serological detection of TBTV.
