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| Cloning, Expression and Purification of Vegetative Insecticidal Protein 3Aa Gene(vip3Aa) from Bacillus thuringiensis WB5 |
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Abstract Vegetative insecticidal proteins(Vips) are one kind of novel toxic proteins, which are generated and excreted by Bacillus thuringiensis(Bt) cells during growth from mid-exponential phase to the stationary phase. Vips may generally be classified into 3 groups of Vip1, Vip2 and Vip3, of which Vip3 is the most intensively studied. It has important research significance because of broad-spectrum insecticidal activities to Lepidoptera pests. The entire coding region of vip3Aa(GenBank No. AAM22456) gene was amplified by PCR with total DNA extracted from BtWB5 as template. PCR product was purified and ligated with pMD18-T to form the recombinant pMD18-T-vip3Aa, and then transformed into Escherichia coli DH5. The recombinant plasmid DNA extracted from the selected positive clone was used for nucleotide sequencing and NdeⅠ/XbaⅠ digested analysis. Using Universal DNA Purification Kit, an approximate 2.4 kb DNA product was purified from NdeⅠ/XbaⅠdigested pMD18-T-vip3Aa, and then inserted into multiple cloning sites of the expression vector pCzn1 to generate the recombinant expression vector pCzn1-vip3Aa. The inserted fragment and its reading frame were confirmed by NdeⅠ/XbaⅠdigestion and nucleotide sequence analysis. pCzn1-vip3Aa was transformed into E. coli ArcticExpressTM (DE3) and induced with isopropyl β-D-1-thiogalactopyranoside(IPTG). SDS-PAGE showed that the relative molecular weight of the expressed protein was about 88 kD in both supernatant and precipitated. In addition, the expressed protein was purified by Ni-NTA affinity chromatography. The success of the subcloned vip3Aa gene and the expression and purification of Vip3Aa provide basic data for furthur research on its action receptor in insect mid gut.
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Received: 03 June 2014
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NI He-Jia, CHENG Xue, WANG Jing, GAO Ze-Ping, WEI Pan-Pan, TAN Jia-Li, SUN Jing, LI Hai-Tao, GAO Ji-Guo. Isolation and Screening of High Virulent Bacillus thuringiensis Strains Against Lepidopteran Pests in Some Areas of Liaoning Province[J]. 农业生物技术学报, 2019, 27(8): 1513-1520. |
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