关键词: APN, 杆状病毒表达载体, Tn, 表达

Abstract: 【Objective】To successfully express the Bt (Bacillus thuringiensis) toxin receptor protein located on internal membrane of larval midgut of cotton blooworm (Helicoverpa armigera Berliner), within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance.【Method】By using PCR method to separately amplify the fragments without signal peptide of Aminopeptidase N1 gene from midguts of susceptible and resistant H. armigera, and separately cloned into pUC 19 vector. After sequencing the gene, the fragment encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into E scherichia coli DH10Bac. It was cultured in LB plate which contained Te, Kan, Ge, X-gal and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni (Tn) and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot. 【Result】The results showed that the recombinant baculoviruse was fully capable of expressing APN1. 【Conclusion】The APN1 gene was successfully expressed in Tn cell established the base for continue research its function and its relationship of resistance with Bt, and can be used as reference mehthod for farther study the expression and function of other receptor proteins.

Key words: APN, baculovirus expression vector, Tn, expression