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The aim of this article is to successfully express the Bt(Bacillus thuringiensis)toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm(Helicoverpa armigera Hübner)within eukaryotic expression system,which is one of the key links for clarifying the relationship between receptor and Bt resistance.The fragments of aminopeptidase N1(APNI)gene without signal peptide in the susceptible and the resistant H.armigera were cloned separately using PCR method,and were separately cloned into pUC 19 vector.After sequencing the gene,the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter.The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac.It was cultured in LB medium,which contained Te, Kan,Ge,X-gal,and IPTG.The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained.The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis.The results showed that the recombinant baculoviruse was fully capable of expressing APN1.The APN1 gene successfully expressed in T.ni cell established the base for continuing the research on its function and relationship of resistance with Bt.
The aim of this article is to successfully express the Bt (Bacillus thuringiensis) toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm (Helicoverpa armigera Hübner) within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 (APNI) gene without signal peptide in the susceptible and resistant H. hamigera were cloned separately using PCR method, and were cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB / APN1 was screened and transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG.The resulting recombinant bacmid was transfected into cells of t he insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene was successfully expressed in T. ni cell established the base for continuing the research on its function and relationship of resistance with Bt.