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目的构建细粒棘球幼(Eg)14-3-3与MKK2酵母双杂交系统,并对诱饵质粒进行自激活活性及毒性检测。方法从Eg cDNA中扩增Eg14-3-3基因和EgMKK2编码序列,将其克隆入酵母双杂交载体pGADT7和pGBKT7中,经PCR、限制性酶切鉴定及序列测定正确后,PEG/LiAc法转化酵母菌株,检测融合蛋白对酵母菌生长的影响和自激活活性。结果扩增Eg14-3-3和EgMKK2基因编码区全长分别为749bp和1 572bp。构建的pGBKT7-Eg14-3-3及pGADT7-EgMKK2重组质粒转化到酵母细胞Y2HGold中,检测表达蛋白对Y2HGold无毒性及自激活作用。结论 Eg14-3-3基因和EgMKK2编码序列表达的蛋白对酵母菌Y2HGold无毒性和自激活作用,为进一步运用酵母双杂交技术筛选与之相互作用的蛋白奠定了基础。
Objective To construct a yeast two-hybrid system of Echinococcus granulosus (Eg) 14-3-3 and MKK2, and to test the self-activation activity and toxicity of bait plasmid. Methods Eg14-3-3 gene and EgMKK2 coding sequence were amplified from Eg cDNA and cloned into yeast two-hybrid vector pGADT7 and pGBKT7. After PCR, restriction enzyme digestion and sequencing analysis, PEG / LiAc transformation Yeast strains, the detection of the fusion protein on the growth of yeast and self-activation activity. Results The full-length coding regions of Eg14-3-3 and EgMKK2 were 749bp and 1 572bp, respectively. The constructed pGBKT7-Eg14-3-3 and pGADT7-EgMKK2 recombinant plasmids were transformed into yeast cell Y2HGold to detect the non-toxic and self-activating effect of the expressed protein on Y2HGold. Conclusion The non-toxic and self-activating effect of the protein expressed by Eg14-3-3 and EgMKK2 coding sequences on yeast Y2HGold laid the foundation for the further screening of the proteins interacting with yeast two-hybrid system.