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目的构建细粒棘球蚴(Eg)胰岛素受体(InsR)与人胰岛素(Huins)酵母双杂交系统,检测表达蛋白对报告基因的自激活活性及对酵母菌的毒性。方法从Eg cDNA中扩增EgInsR基因和Huins编码序列,分别将EgInsR基因和Huins编码序列克隆入酵母双杂交载体pGADT7和pGBKT7中并进行限制性酶切鉴定和测序鉴定,鉴定正确的重组载体采用PEG/LiAc法转化酵母菌,检测诱饵蛋白对酵母菌生长的自激活活性和毒性。结果扩增的EgInsR和Huins基因编码区全长分别为1 122bp和261bp。构建的pGADT7-EgInsR及pGBKT7-Huins两组重组质粒转化Y2HGold酵母菌表达的蛋白对Y2HGold无毒性及自激活作用。结论构建的EgInsR基因和Huins基因重组载体表达蛋白对Y2HGold酵母菌无毒性和自激活作用,为研究Eg与中间宿主的交互作用机制奠定了基础。
Objective To construct a yeast two - hybrid system (EHR) of human Echinococcus granulosus (Eg) and human insulin (Huins) to detect the self - activation activity of the expressed protein on the reporter gene and its toxicity to yeast. Methods EgInsR gene and Huins coding sequence were amplified from Eg cDNA. EgInsR gene and Huins coding sequence were cloned into yeast two-hybrid vector pGADT7 and pGBKT7, respectively. The restriction enzyme digestion and sequencing were used to identify the correct recombinant vector. / LiAc method was used to transform yeast to test the self-activation activity and toxicity of bait protein on the growth of yeast. Results The full-length coding region of EgInsR and Huins genes were 1 122 bp and 261 bp, respectively. The constructed pGADT7-EgInsR and pGBKT7-Huins recombinant plasmids transformed Y2HGold yeast expression of the non-toxic to Y2HGold and self-activation. CONCLUSION: The constructed recombinant protein of EgInsR and Huins gene are non-toxic and self-activating to Y2HGold yeast, which lays the foundation for studying the interaction between Eg and intermediate host.