细粒棘球蚴MKK1和MKK2基因原核表达载体的构建及其诱导表达  

Construction and prokaryotic expression of the MKK1 and MKK2 genes of Echinococcus granulosus

作　　者：张传山[1] 杨乐[1] 王丽敏[1] 李亮[1] 王俊华[1] 吕国栋[1] 林仁勇[1] 

机构地区：[1]新疆医科大学第一附属医院、新疆重大疾病医学重点实验室一省部共建国家重点实验室培育基地、新疆包虫病基础医学重点实验室,新疆乌鲁木齐830054

出　　处：《中国病原生物学杂志》2014年第1期43-47,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81101271,81260252);长江学者和创新团队发展计划项目(No.IRT1181);新疆医学动物模型研究重点实验室开放课题(No.XJDX1103-2012-03)

摘　　要：目的分别构建细粒棘球蚴(Eg)丝裂原活化蛋白激酶激酶MKK1和MKK2基因原核表达载体,诱导表达并纯化EgMKK1和EgMKK2蛋白。方法采集绵羊肝脏感染的Eg原头蚴,Trizol法提取原头蚴总RNA,反转录PCR分别扩增EgMKK1和EgMKK2基因片段,克隆至原核表达载体pET28a(+),经限制性内切酶双酶切和测序鉴定正确后转化大肠埃希菌感受态细胞BL21,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,采用镍柱亲和层析法纯化蛋白,并进行SDS-PAGE分析。结果测序证明pET28a-EgMKK1和pET28a-EgMKK2原核表达载体构建正确,在37℃经IPTG(终浓度1mmol/L)诱导可表达EgMKK1和EgMKK2蛋白,分子质量单位分别为44.96ku和64.01ku,与理论值相符,且以包涵体形式存在。表达产物纯化后的可溶性蛋白EgMKK1和EgMKK2含量分别为0.64mg/ml和1.2mg/ml。结论成功构建了MKK1和MKK2基因原核表达载体,表达蛋白可用于动物免疫,为研究EgMKK1和EgMKK2在Eg体内的生物学作用奠定了基础。Objectives To construction prokaryotic expression vectors to express mitogen-activated protein kinases （MKK1 and MKK2） of Echinococcus granulosus and to express and purify EgMKK1 and EgMKK2 proteins. Methods TRIzol was used to extract total RNA from livers of sheep infected with E. granulosus. EgMKK1 and EgMKK20RFs were amplified with reverse transcription PCR and then cloned into the prokaryotic expression vector pET28a （T）. After identification with restriction endonucleases and sequence determination, the correct recombinant vector was transferred into Escherichia coli BL21 competent cells. Different concentrations of IPTG were used to induce the expression of EgMKK1 and EgMKK2 fusion proteins. Protein was purified using Ni2＋ affinity chromatography, and the purity of the recombinant protein was tested using SDS-PAGE. Results The prokaryotic expression vectors pET28aEgMKK1 and pET28a-EgMKK2 were constructed correctly, and IPTG （final concentration 1 mmol/L） induced the expression of the fu- sion proteins EgMKK1 and EgMKK2 at 37 ℃. The fusion proteins EgMKK1 and EgMKK2 had a respective molecular mass of 44.96 ku and 64.01 ku. The fusion proteins were successfully purified using a Ni-NTA purification column. The purified soluble proteins EgMKK1 and EgMKK2 were 0. 64 mg/ml and 1.2 mg/ml. Conclusion The purified fusion proteins EgMKK1 and EgMKK2 can be used in animal immunization. This work has laid the foundation for further re- search on the biological role of EgMKK1 and EgMKK2 in vivo.

关 键 词：细粒棘球绦虫 MKK1和MKK2基因 基因表达 蛋白纯化 

分 类 号：R383.33[医药卫生—医学寄生虫学]