论文部分内容阅读
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1μg RNA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BamHⅠ、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/LiAc法将重组载体转化入感受态酵母菌株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1 017bp、1 086bp和1 107bp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Y2HGold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。
Objective To construct Eukaryotic expression vector of EgMKK1 and its downstream members ERK and P38 yeast two-hybrid vector of Echinococcus granulosus (Eg) and to detect the toxic effect and self-activation activity of the fusion protein on the growth of yeast . Methods Eg prototheca infection in sheep liver was harvested five times with normal saline. The total RNA was extracted by TRIzol method and 1 μg RNA was reverse transcribed into cDNA. The EgMKK1, EgERK and EgP38 gene fragments were amplified by PCR and cloned into pGADT7 or pGADT7 by restriction endonucleases EcoRⅠand BamHⅠ, EcoRⅠand BamHⅠ, EcoRⅠand SalⅠ, respectively. pGBKT7 was identified by restriction enzyme digestion and sequencing. The recombinant vector was transformed into competent yeast strain Y2HGold by PEG / LiAc method. The recombinant plasmid pGBKT7 was screened by solid medium to detect the toxic effect and self-activation activity of the fusion protein on the growth of the yeast strain. Results The target genes EgMKK1, EgERK and EgP38 were obtained by PCR amplification. The constructed recombinant yeast two-hybrid vectors pGADT7-EgMKK1, pGBKT7-EgERK and pGBKT7-EgP38 were double digested to obtain the target gene fragments of 1 017 bp, 1 086 bp and 1 107 bp, respectively, and the sizes were consistent with the predicted values. Recombinant yeast two-hybrid vector transformed Y2HGold yeast on SD / -Trp plate diameter of 1.5 ~ 2.0mm colonies, and the original vector pGADT7 or pGBKT7 transformed yeast colonies the same size; recombinant yeast two-hybrid vector transformed yeast in SD / Both the -Trp and SD / -Leu plates grew white colonies about 2 mm in diameter, whereas blue colonies did not grow on the SDO / X / A and DDO / X / A solid medium plates. Conclusion The yeast two-hybrid vectors pGADT7-EgMKK1, pGBKT7-EgERK and pGBKT7-EgP38 were successfully constructed and their expressed proteins were non-toxic and self-activating to yeast Y2HGold. To further investigate the interaction between EgMKK1 and its downstream EgERK and EgP38 The foundation.