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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: To clone the cry3Aa gene into AcNPV Polyhedrin gene and construct a recombinant prokaryotic expression vector and express it in E. coli. Methods: Firstly, cry3Aa gene was amplified by PCR using plasmid DNA of PHT305a as a template. Polyhedrin gene was amplified by PCR using PET30a-ph-1a plasmid DNA as a template. The two gene fragments were respectively linked into PMD18-T cloning vector and sequenced and identified. The construction of the expression vector was divided into two steps: firstly, the cry3Aa gene cut by SacI and XhoI double enzymes was ligated into the expression vector pET30a, then the PMD18T-Ph was digested by BglII and SacI to obtain the ph gene fragment with BglII / The pET30a-cry3Aa was ligated with the SacI end to construct the expression vector pET30a-cry3Aa-ph. The expression vector was transformed into E. coli BL21 (DE3). IPTG induced protein expression and purification, SDS-PAGE analysis of the results. Results: The sequences of cry3Aa and AcNPV Polyhedrin genes were identical with those reported. The foreign proteins expressed by 1mmol / L IPTG for 4h showed the specific bands of expressed products at about 103kDa by SDS-PAGE. Conclusion: The recombinant protein engineering strain with cry3Aa and polyhedrine genes laid the foundation for the application of this gene in insecticides.