手掌参γ-硫素的原核表达及纯化  

Prokaryotic Expression and Purification of γ-thionin of Gymanadenia conopsea R.Br.

作　　者：李雪峰[1] 周建平[1] 刘艳[1] 袁剑波[1] 郝军莉[1] 冯娟[1] 任正隆[1] 

机构地区：[1]电子科技大学生命科学与技术学院,四川成都610054

出　　处：《生物技术通讯》2009年第2期187-190,共4页Letters in Biotechnology

基　　金：国家自然科学基金重点项目(30730065)

摘　　要：目的:在原核系统中高效表达手掌参γ-硫素,并对其进行纯化。方法:通过筛选手掌参cDNA文库获得γ-硫素基因(gcthionin),分别对其全长及信号肽编码序列缺失的cDNA片段进行PCR扩增,克隆入原核表达载体pET-32(a),构建重组质粒pET-32(a)/gcthionin和pET-32(a)/Δgcthionin;测序鉴定后,转化大肠杆菌BL21(DE3),经IPTG诱导表达融合蛋白;SDS-PAGE分析后,采用Ni-NTA亲和层析柱及凝胶柱对可溶性蛋白进行纯化,Western blotting鉴定。结果:gcthionin基因开放式阅读框全长225nt,编码一个由74个氨基酸残基组成的蛋白;带有信号肽的重组质粒在大肠杆菌BL21(DE3)中以包涵体形式表达;信号肽缺失可以极大地提高外源蛋白的可溶性,该可溶性产物经Ni-NTA柱及凝胶过滤后可获得纯度较高的蛋白,经Western blotting分析,相对分子质量约21.9×103处有明显的蛋白条带,与预期蛋白分子大小一致。结论:信号肽编码序列缺失的Δgcthionin可在大肠杆菌中可溶、高效表达。Objective： To express γ-thionin of Gymandenia conopsea R.Br. in prokaryotic system and purify it. Methods： The γ-thionin gene（gcthionin） was screened from the cDNA library of young shoot of G.conopsea R.Br. The fulllength of ORF and the signal peptide-truncated fragment were amplified and cloned into the pET-32（a） vector respectively. The two resulting plasmids pET-32（a）/gcthionin and pET-32（a）/Agethionin were identified by DNA sequencing and transformed into E.coli BL21（DE3） to express the fusion protein by the induction of IPTG. After analysis of the solubility, the expressed soluble protein was further purified by Ni-NTA column and gel filtration chromatography, and then was identified by Western blotting. Results： ORF of gcthionin gene was 225 nt, encoding 74 amino acid residues. It was found that the presence of signal peptide contributed to the formation of inclusion body, and the absence of signal peptide greatly enhanced the solubility of the heterogenous protein. Further purification of the deleted protein was achieved using Ni-NTA column and gel filtration chromatography. Result of Western blotting showed that one distinct band around the expected molecular weight of 21.9 kD. Conclusion： The gene fragement Agcthionin without signal peptide could be expressed in E. coli system highly and in a soluble form. This study will be very fundamental and essential for further investigation on the stucture and function of gcthionin.

关 键 词：手掌参 γ-硫素 原核表达 

分 类 号：Q78[生物学—分子生物学]