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| Expression of Vegetative Insecticidal Protein 3Aa Gene (vip3Aa) from Bacillus thuringiensis NBIC-380 in Escherichia coli and Bacillus Strains |
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Abstract Vegetative insecticidal protein 3A (Vip3A) has a broad specturm toxicity torwards Lepidoptera pests. The 2.4 kb entire coding region of vip3Aa gene (GenBank No. KT307982) was amplified by PCR with total DNA extracted from Bacillus thuringienesis (Bt) NBIC-380 which had high insecticidal activity against corn borer (Ostrinia nubilalis) as template. The vip3Aa was inserted into multiple cloning sites of the Escherichia coli expression vector pGEX-6p-1 and pET-28a, Bacillus expression vector pBMB1A to generate the recombinant expression vector pGV3, pEV3 and pBV3. The pGV3 and pEV3 were transformed into E. coli BL21 (DE3), respectively, and pBV3 was electro- transformed into Bt BMB171 and Bacillus subtilis (Bs) 168 strains. Sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) showed one protein of 88 kD. The expressed protein in E. coli was purified by GST affinity chromatography and Ni affinity chromatography, which demonstrated no toxicity towards Helicoverpa armigera second instar larvae. The proteins expressed in BtBMB171 and Bs168 had activities against H. armigera second instar larvae with medium lethal concentration (LC50) of 6.185 6 and 2.984 6 mg/mL, respectively. Bioassay indicated that expression of vip3Aa gene from Bt NBIC-380 strain had certain toxicity torwards H. armigera. This work enriches the fundamental research of Vip and provides basic data for construction of high toxicity and broad spectrum engineering strains.
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Received: 01 April 2015
Published: 05 October 2015
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