摘要
昆虫GABA受体(γ-aminobutyric acid receptor,GABAR)是杀虫剂的重要靶标之一。本研究以黑腹果蝇Drosophila melanogaster整体组织的cDNA作为模板,采用RT-PCR技术扩增了黑腹果蝇GABA受体LCCH3亚基和GRD亚基的cDNA序列,并克隆至pET-32a表达载体上,测序结果表明获得的序列与基因库中已发表的序列一致性在99%以上,无移码突变。在IPTG的诱导下,LCCH3基因成功在大肠杆菌Escherichia coli中表达,而GRD基因未表达。通过包涵体洗涤、变性、Ni2+亲合层析纯化、稀释复性获得纯化的重组表达的LCCH3蛋白,并用圆二色谱测定了目标蛋白的二级结构,主要富含β结构。该研究结果为研究昆虫GABAR的结构和功能关系提供了重要的参考数据。
γ-Aminobutyric acid receptor（GABAR） is one of the most important insecticide targets.In this study,the fragments of genes of GRD and LCCH3 subunits in Drosophila melanogaster were amplified by RT-PCR and cloned into pET-32a expression vector.Homologous analysis showed that they have more than 99% identity with the published amino acid sequences of the homologous genes in GenBank,without frameshift mutation.The recombinant plasmids were transformed into Escherichia coli and expressed under IPTG induction.LCCH3 gene was expressed successfully,but GRD gene failed.The expressed LCCH3 subunit was purified by the processes of inclusion body washing,denaturation,Ni2＋ affinity chromatography and renaturation.The secondary structure of LCCH3 subunit was determined by circular dichroism（CD） spectroscopy,the result showed that this subunit was rich in β-strand.The results provide important information for the study of relationship between the structure and function of GABA receptor.
出处
《昆虫学报》
CAS
CSCD
北大核心
2012年第3期259-266,共8页
Acta Entomologica Sinica
基金
国家自然科学基金项目(20872093,21172147)
上海市教委基金项目(11ZZ122)
