农业生物技术学报
農業生物技術學報
농업생물기술학보
JOURNAL OF AGRICULTURAL BIOTECHNOLOGY
2014年
11期
1388-1393
,共6页
陈凡冰%宋飞飞%石鹏%黄志鹏%关雄
陳凡冰%宋飛飛%石鵬%黃誌鵬%關雄
진범빙%송비비%석붕%황지붕%관웅
苏云金芽胞杆菌%营养期杀虫蛋白3Aa基因(vip3Aa)%克隆与表达%蛋白纯化
囌雲金芽胞桿菌%營養期殺蟲蛋白3Aa基因(vip3Aa)%剋隆與錶達%蛋白純化
소운금아포간균%영양기살충단백3Aa기인(vip3Aa)%극륭여표체%단백순화
Bacillus thuringiensis%Vegetative insecticidal proteins 3Aa gene(vip3Aa)%Cloning and expression%Protein purification
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。
營養期殺蟲蛋白(vegetative insectcidal protein, Vip)是由囌雲金芽胞桿菌(Bacillus thuringiensis, Bt)在營養生長指數中期至穩定期期間分泌產生的一類新型殺蟲因子，分為Vip1、Vip2和Vip3三種，以Vip3的研究最為深入。Vip3A對鱗翅目(Lepidoptera)害蟲具有廣譜殺蟲活性，具有重要研究意義。本研究以BtWB5菌株總DNA為模闆，採用PCR方法擴增vip3Aa(GenBank登錄號：AAM22456)全長，將該片段純化迴收後與pMD18-T連接併轉入大腸桿菌(Escherichia coli)DH5α，進行酶切驗證和序列測定。將vip3Aa基因片段與經同樣酶切的錶達載體pCzn1連接，構建重組錶達載體pCzn1-vip3Aa，轉入大腸桿菌ArcticExpressTM (DE3)，異丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside, IPTG)進行誘導錶達。SDS-PAGE結果錶明，在沉澱和上清中均有約88 kD VIP3Aa蛋白質錶達產物；採用Ni親和樹脂對上清中的VIP3Aa融閤錶達蛋白進行瞭純化，穫得瞭純化蛋白。本研究成功亞剋隆瞭vip3Aa基因，併錶達純化瞭VIP3Aa蛋白，為後續研究VIP3Aa蛋白在昆蟲中腸的作用受體提供瞭基礎資料。
영양기살충단백(vegetative insectcidal protein, Vip)시유소운금아포간균(Bacillus thuringiensis, Bt)재영양생장지수중기지은정기기간분비산생적일류신형살충인자，분위Vip1、Vip2화Vip3삼충，이Vip3적연구최위심입。Vip3A대린시목(Lepidoptera)해충구유엄보살충활성，구유중요연구의의。본연구이BtWB5균주총DNA위모판，채용PCR방법확증vip3Aa(GenBank등록호：AAM22456)전장，장해편단순화회수후여pMD18-T련접병전입대장간균(Escherichia coli)DH5α，진행매절험증화서렬측정。장vip3Aa기인편단여경동양매절적표체재체pCzn1련접，구건중조표체재체pCzn1-vip3Aa，전입대장간균ArcticExpressTM (DE3)，이병기-β-D-류대필남반유당감(isopropylβ-D-1-thiogalactopyranoside, IPTG)진행유도표체。SDS-PAGE결과표명，재침정화상청중균유약88 kD VIP3Aa단백질표체산물；채용Ni친화수지대상청중적VIP3Aa융합표체단백진행료순화，획득료순화단백。본연구성공아극륭료vip3Aa기인，병표체순화료VIP3Aa단백，위후속연구VIP3Aa단백재곤충중장적작용수체제공료기출자료。
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.
