AcNPV polyhedrin与cry3Aa基因的重组与重组融合蛋白在大肠菌中的表达

Recombination of cry3Aa and AcNPV Polyhedrine and Preliminary Expression of the Recombined Fusion Protein in E.coli

目的:将cry3Aa基因与斜纹夜蛾多角体蛋白(AcNPV Polyhedrin)基因重组,构建重组蛋白原核表达载体并在大肠杆菌中表达。方法:首先以PHT305a载体质粒DNA为模板,PCR扩增cry3Aa基因。再以PET30a-ph-1a载体质粒DNA为模板,PCR扩增多角体蛋白基因。将两基因片段分别连入PMD18-T克隆载体,测序鉴定。表达载体的构建分为两步,先将SacI和XhoI双酶切下来的cry3Aa基因连入表达载体pET30a,再将PMD18T-Ph经BglⅡ和SacI双酶切,所获得的ph基因片段与具有BglⅡ/SacI末端的pET30a-cry3Aa相连接,构建表达载体pET30a-cry3Aa-ph,表达载体转化进入大肠杆菌BL21(DE3)。IPTG诱导蛋白表达并纯化,SDS-PAGE分析结果。结果:cry3Aa和AcNPV Polyhedrin基因序列与报道完全一致。1mmol/LIPTG诱导4h表达的外源蛋白,经SDS-PAGE分析显示在大约103kDa处有表达产物特异条带。结论:获得cry3Aa与polyhedrine基因的重组蛋白工程菌株,为此基因应用于杀虫剂奠定了基础。

Objective:Recombined cry3Aa with AcNPV Polyhedrin,constuct the express vector and made the recombined protein expressed in E.coli.Method;First,cry3Aa gene was amplified from vector PHT305a.Second,Polyhedrin gene was amplified from vector PET30a-ph-l a.Two DNA fragments were ligased intoPMD18-T vector respectively and sequenced the two vectors.For construction of the protein expression vector PET30a-cry3Aa-ph:first,cry3Aa fragment from PMD18T-cry3Aa SacI/XhoI endonuclease digestions was ligased with pET-30a,and then Ph from PMD18T-Ph SacI/XhoI endonuclease digestions was inserted into pET30a-cry3Aa.The vector PET30a-cry3Aa-ph was transducted into BL21(DE3) to express the recombinant protein.Purified fusion protein was obtained through ultrasonic fragmentation and purification using inclusion body washing buffer,and then analyzed with SDS-PAGE.Result:Sequencing results showed that sequences of the DNA of cry3Aa and polh are consist with the sequence reported.Result of SDS-PAGE analyzation revealed that there was a band of exprssion product at 103kDa.Conclusion:The recombined protein engineering strain of cry3Aa and AcNPV Polyhedrin was obtained,which laid foundation for the application in pesticide.