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梨小食心虫普通气味结合蛋白2(GmolGOBP2)cDNA的克隆与原核表达
张国辉1,2, 仵均祥1
1.西北农林科技大学 植物保护学院 植保资源与病虫害治理教育部重点实验室,应用昆虫学重点实验室;2.长江大学 农学院 昆虫研究所,湿地生态与农业利用教育部工程研究中心
摘要:
【目的】克隆梨小食心虫(Grapholita molesta)普通气味结合蛋白2 (GmolGOBP2)的全长cDNA序列并进行原核表达, 为研究该蛋白在梨小食心虫化学感受系统中的作用奠定基础。【方法】利用RT-PCR和RACE技术克隆GmolGOBP2的全长cDNA序列,并使用原核表达载体pET-32a在大肠杆菌BL21(DE3)中进行表达,用SDS-PAGE和Western blot检测其表达情况。【结果】GmolGOBP2的cDNA全长序列为 637 bp(GenBank登录号:JN857940),开放性阅读框长度为483 bp,编码161个氨基酸残基,成熟蛋白分子质量为15.98 ku,等电点为4.85。GmolGOBP2预测蛋白的N末端具20个氨基酸残基组成的信号肽序列,并且氨基酸序列中具有6个保守半胱氨酸的典型气味结合蛋白家族标志。将GmolGOBP2编码序列重组到表达载体pET-32a中,转入大肠杆菌BL21(DE3)进行原核表达,SDS-PAGE和Western blot检测结果显示,梨小食心虫普通气味结合蛋白基因在大肠杆菌中成功地表达出分子质量约为32 ku的融合蛋白,与预测的融合蛋白分子质量大小一致。【结论】克隆并原核表达了GmolGOBP2 的cDNA序列,可用于研究该蛋白分子结构及其在化学感受系统中的功能。
关键词:  梨小食心虫  普通气味结合蛋白2  分子克隆  原核表达
DOI:
分类号:
基金项目:国家自然科学基金项目(31501641,31272043);农业部公益性行业(农业)科研专项(201103024)
Cloning and prokaryotic expression of general odorant binding protein 2 cDNA from oriental fruit moth,Grapholita molesta
ZHANG Guohui,WU Junxiang
Abstract:
【Objective】Cloning and prokaryotic expression of a novel cDNA encoding the general odorant binding protein 2 (GOBP2) from the oriental fruit moth Grapholita molesta was conducted.【Method】The full-length cDNA encoding GOBP2 isolated from Grapholita molesta by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends-PCR (RACE PCR) was named as GmolGOBP2.It was then constructed into the expression vector pET-32a and expressed in Escherichia coli BL21 (DE3).【Result】The full length cDNA of GmolGOBP2 was 637 bp (GenBank accession no.JN857940),containing a 483 bp open reading frame with 161 amino acids.The deduced molecular weight (MW) was 15.98 ku and the PI was 4.85.Protein signature analysis indicated that the deduced GmolGOBP2 contained an N-terminal signal sequence of 20 amino acids.The GmolGOBP2 was then constructed into the expression vector pET-32a and expressed in Escherichia coli BL21 (DE3) after induction with IPTG.SDS-PAGE and Western blot analysis showed the molecular weight of the recombinant GmolGOBP2 was about 32 ku,in consistence with the predicted result.【Conclusion】GmolGOBP2 was cloned and expressed in prokaryotic expression system,which was helpful for further studies on its molecular structure and function in the olfactory system.
Key words:  Grapholita molesta  general odorant binding protein 2  molecular cloning  prokaryotic expression