AMP激活的蛋白激酶α2大肠杆菌双杂交诱饵重组质粒的构建及鉴定

Construction and identification of bait plasmid carrying AMP-activated protein kinase α2 in bacterial two-hybrid system

目的构建大肠杆菌双杂交系统的诱饵重组质粒。方法PCR扩增大鼠AMP激活的蛋白激酶α2(AMP-ac-tivated prote in k inaseα2,AMPKα2)蛋白编码区cDNA,与大肠杆菌双杂交表达载体pBT构建融合蛋白表达质粒pBT-AMPKα2。经酶切和测序鉴定后,将pBT-AMPKα2转化XL-1 B lue MRF’大肠杆菌报告菌株,检测诱饵融合蛋白的表达。并用pBT-AMPKα2和pTRG空载体共转化XL-1 B lue MRF’菌株,通过3-氨基-1,2,4-三唑(3-am ino-1,2,4-triazole,3-AT)选择性筛选平板检测诱饵融合蛋白的自激活作用。结果酶切和测序结果表明AMPKα2蛋白编码序列成功克隆入pBT载体,融合区阅读框正确。诱饵重组质粒pBT-AMPKα2能表达λcI/AMPKα2融合蛋白。转化有pBT-AMPKα2和pTRG空载体的XL-1 B lue MRF’大肠杆菌报告菌株在3-AT选择性筛选平板上不生长,而在no 3-AT非选择性筛选平板上生长良好,表明pBT-AMPKα2重组质粒能在大肠杆菌细胞中正确表达且无自激活作用。结论构建的pBT-AMPKα2诱饵重组质粒可用于下一阶段的cDNA文库筛选。

Objective To construct a bait plasmid in bacterial two-hybrid system. Methods A cDNA fragment encoding for rat AMP-activated protein kinase α2 （AMPKα2） was amplified by PCR and inserted into bacterial expression vector pBT. After confirmation with restricted endonuclease digestion and sequence analysis, bacterial reporter strain XL-1 Blue MRF＇ was transformed with pBT-AMPKα2 plasmid and the expression of the recombinant bait fusion protein was detected. To test whether the bait fusion protein had the capability of self-activation, the XL-1 Blue MRF＇ cells were cotransformed with the pBT-AMPKα2 plasmid and empty pTRG vector, and screened on 3-amino-1,2,4-triazole （3-AT） Selective Screening Medium plates. Results Restriction digestion and sequence analysis revealed that the AMPKα2 code sequence was correctly inserted into pBT with a right reading frame, pBT-AMPKα2 expressed λcI/AMPKα2 fusion protein. Colonies were obtained on no 3-AT Nonselective Screening Medium plates when XL-1 Blue MRF＇ cells were cotransformed with recombinant pBT-AMPKα2 and empty pTRG vector, while none grew on 3-AT plates, indicating that the recombinant plasmid pBT-AMPKα2 expressed AMPKα2/λcI fusion protein correctly, and was incapable of activation of the reporter cassette in the absence of an interaction partner. Conclusion The recombinant plasmid pBT- AMPKα2 could be used as ＂bait plasmid＂ to screen cDNA library.