细粒棘球蚴MAPKK样激酶及其下游成员ERK和P38酵母双杂交载体的构建与自激活鉴定  

Construction of a yeast two-hybrid system with Echinococcus granulosus MAPKK-like kinase and its downstream kinases ERK and P38 and testing of its autoactivation

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3期207-210,215,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：目的构建细粒棘球蚴（Echinococcus granulosus，Eg）丝裂原活化蛋白激酶激酶（EgMKK1）及其下游成员ERK、P38酵母双杂交载体，并检测融合蛋白对酵母菌生长的毒性作用及自激活活性。方法采集绵羊肝脏感染的Eg原头蚴，生理盐水冲洗5次，TRIzol法提取原头蚴总RNA，取1ugRNA反转录cDNA。以cDNA为模板，PCR分别扩增EgMKK1、EgERK和EgP38基因片段，分别经限制性内切酶EcoRI和BamHI、EcoRI和BarnHI、EcoRI和SalI双酶切，酶切片段连接至酵母双杂交载体pGADT7或pGBKT7，经酶切及测序鉴定正确后，应用PEG／I。iAc法将重组载体转化人感受态酵母菌株Y2HGold，利用固体培养基筛选，检测融合蛋白对酵母菌株生长的毒性作用及自激活活性。结果经PCR扩增，分别获得目的基因EgMKK1、EgERK和EgP38。构建的重组酵母双杂交载体pGADT7-EgMKK1、pGBKT7-EgERK及pGBKT7-EgP38经双酶切，分别得到1017bp、1086bp和1107bp目的基因片段，大小与预测值相符。重组酵母双杂交载体转化Y2HG01d酵母菌在SD／-Trp平板上形成直径为1．5～2．0mm的菌落，与原始载体pGADT7或pGBKT7转化酵母菌菌落大小一致；重组酵母双杂交载体转化酵母菌在SD／-Trp和SD／-Leu平板均有直径约2mm白色菌落生长，而SDO／X／A和DDO／x／A固体培养基平板上无蓝色菌落生长。结论成功构建了酵母双杂交载体pGADT7-EgMKK1、pGBKT7-EgERK和pGBKT7-EgP38，其表达蛋白对酵母菌Y2HGold无毒性和自激活作用，为进一步研究EgMKK1与其下游成员EgERK、EgP38之间的相互作用奠定了基础。Objectives To construct a yeast two-hybrid system with Echinococcus granulosus mitogen-activated protein kinase kinase （EgMKK1） and its downstream kinases ERK and P38 and to detect the fusion protein＇s toxicity to yeast growth and its autoactivation. Methods E. granulosus was isolated from livers of sheep infected with E. granulosus protoscoleces. Livers were rinsed 5 times with physiological saline. Total RNA was extracted from E. granulosus protoscoleces using TRIzol, and then 1 ug EgRNA was reverse transcribed to eDNA. EgMKK1, EgERK, and EgP38 gene fragments from EgRNA were amplified using PCR and digested with the respective restriction endonucleases EcoR I and BamH I , EcoR I and BamH I , and EcoR I and Sal I . The coding sequence was then cloned into the yeast two-hybrid vectors pGADT7 and pGBKT7. After identification using PCR, restriction analysis, and sequencing, the recombinant vector was transformed into the yeast strain Y2HGold using the PEG/LiAc method to detect its toxicity and determine its autoactivation using solid medium screening. Results The target genes EgMKK1, EgERK, and Eg p38 were obtained using PCR amplification. After the recombinant yeast two-hybrid vectors were digested with restriction enzymes, target gene fragments of 1 017 bp, 1 086 bp and 1 107 bp were obtained, and these sizes were consistent with predicted values. The Y2HGold yeast strain containing the recombinant yeast two-hybrid vector grew well on SD/-Trp plates with a colony size of 1.5--2.0 mm, which was similar to the colony size for the positive control. Yeast containing the recombinant yeast two-hybrid vectors produced white colonies about 2 mm in size on SD/-Trp and SD/-Leu plates. There was no growth of blue colonies on SDO/X/A and DDO/X/A plates. Caaelasian Yeast two-hybrid vectors of EgMKK1, EgERK and EgP38 were successfully constructed and lacked toxicity and autoactivation. This lays the foundations for study of the interaction between EgMKK1 and its downstream effectors EgERK and EgP38.

关 键 词：细粒棘球绦虫 MAPK 酵母双杂交 毒性 自激活 

分 类 号：R383.33[医药卫生—医学寄生虫学]