摘要
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 Echinococcus granulosus （Eg）14-3-3 and MKK2 vectors for a yeast two- hybrid sys- tem and test the bait vectors for transcriptional activation and toxicity. Methods Sequences coding for EgMKK2 and Eg14-3-3 were amplified from Eg cDNA by PCR and then subcloned into the yeast two-hybrid vectors pGADT7 and pGBKT7. After verification with PCR, restriction analysis, and sequencing, the recombined plasmids were transformed into a yeast strain by the PEG/LiAc method and their toxicity and self-activation were detected. Results Full-length sequences coding for Eg14-3-3 and EgMKK2 were 749 bp and 1 572 bp, respectively, pGBKT7-Egl4-3-3 and pGADT7EgMKK2 vectors were constructed and found to have no toxicity or self-activation. Conclusion The yeast two-hybrid expression vectors Eg14-3-3 and EgMKK2 were constructed and found to have no toxicity or self-activation, providing a basis for the screening proteins interacting with Eg14-3-3 and EgMKK2 using the yeast two-hybrid technioue.
出处
《中国病原生物学杂志》
CSCD
2011年第12期894-897,F0002,共5页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.30960341
30860253)
新疆维吾尔自治区包虫病基础医学重点实验室开放课题(No.XJDX0202-2010-06)
