营养期杀虫蛋白(vegetative insectcidal protein, Vip)是由苏云金芽胞杆菌(Bacillus thuringiensis, Bt)在营养生长指数中期至稳定期期间分泌产生的一类新型杀虫因子，分为Vip1、Vip2和Vip3三种，以Vip3的研究最为深入。Vip3A对鳞翅目(Lepidoptera)害虫具有广谱杀虫活性，具有重要研究意义。本研究以BtWB5菌株总DNA为模板，采用PCR方法扩增vip3Aa(GenBank登录号：AAM22456)全长，将该片段纯化回收后与pMD18-T连接并转入大肠杆菌(Escherichia coli)DH5α，进行酶切验证和序列测定。将vip3Aa基因片段与经同样酶切的表达载体pCzn1连接，构建重组表达载体pCzn1-vip3Aa，转入大肠杆菌ArcticExpressTM (DE3)，异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)进行诱导表达。SDS-PAGE结果表明，在沉淀和上清中均有约88 kD VIP3Aa蛋白质表达产物；采用Ni亲和树脂对上清中的VIP3Aa融合表达蛋白进行了纯化，获得了纯化蛋白。本研究成功亚克隆了vip3Aa基因，并表达纯化了VIP3Aa蛋白，为后续研究VIP3Aa蛋白在昆虫中肠的作用受体提供了基础资料。%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/XbaⅠdigested 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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