关键词: 野桑蚕, 谷胱甘肽S-转移酶基因, 克隆, 表达, 杆状病毒表达系统, Sf9细胞

Abstract: Glutathione S-transferases (GSTs) were a class of the important xenobiotics metabolizing enzymes in insects. In this research, a 771 bp GST-Omega1 gene (encoded 256 amino acids) was cloned by RT-PCR from the midgut of Bombyx mandarina for exploring the expression profiling of enzyme GSTs in eukaryotic expression system. Analysis by Conserved Domains online tool of NCBI website indicated that the deduced amino acid sequence was comprised of the omega family conserved sequences with one Cys residue and eight GSH binding sites. GST-Omega1 was then cloned into the pFastBacHT b vector, and the recombinant vector was transformed into DH10Bac competent cells to obtain the bacmid DNA. Spodoptera frugiperda Sf9 cells were transfected with the complexes of bacmid DNA and lipofectin to get recombinant baculovirus. The SDS-PAGE and Western blotting results of the expression of recombinant protein showed that a specific band was detected around 33 kD, consistent with the expected size of the fusion protein. The purified target protein by His-Bind resin could catalyze the universal GST substrate CDNB, and exhibited enzymatic activity K_m 2.81 µmol/L and V_max 2.70 μmol/(mg·min).

Key words: Bombyx mandarina, glutathione S-transferases gene, cloning, expression, baculovirus expression system, Sf9 cell