人巨细胞病毒UL123基因外显子4诱饵质粒的构建与鉴定

Construction and validation of human cytomegalovirus UL123 gene exon4 bait plasmid

目的构建和鉴定人巨细胞病毒(HCMV)UL123基因外显子4(ie1-exon4)的诱饵质粒,并评价其用于细菌双杂交系统筛选文库的可行性。方法以重组质粒pTWIN1/ie1为模板扩增ie1-exon4,克隆入pBT质粒,转化E.coliXL1-BlueMRF’Kan,经PCR、限制性酶切和测序鉴定阳性重组子,提取阳性重组质粒,再转化入细菌双杂交系统报告菌株,诱导表达重组融合蛋白,用SDS-PAGE和WesternBlot对表达产物进行鉴定,并进一步鉴定重组子自身激活作用。结果细菌双杂交诱饵质粒pBT/ie1-exon4构建成功,并在报告菌株中表达了重组融合蛋白rIE1-C86-491/λC1,且pBT/ie1-exon4无自身激活作用。结论成功构建了无自激活作用的诱饵质粒pBT/ie1-exon4,可用于筛选文库。

【Objective】 To construct and validate HCMV UL123 gene exon4 （ie1-exon4） bait plasmid, and identify its selfactivation property. 【Methods】HCMV ie1-exon4 carried on pTWIN1/ie1 recombinant was amplified by PCR and cloned into the pBT plasmid, which was transformed into E.coli XL1-Blue MRF＇Kan host strain, and the positive recombinant was identified by PCR, restriction and sequencing analysis. Then the verified positive recombinant was transformed into bacterial two-hybrid system reporter strain. The soluble fusion protein rIE1-C86491/λC1 was expressed, and analyzed by SDS-PAGE and Western blot. Finally, whether the recombinant was able to activate reporter gene by himself or not was identified. 【Results】Bacterial two-hybrid pBT/ie1-exon4 bait plasmid was successfully constructed, and the corresponding fusion protein rIE1-C86-491/λC1 was expressed in bacterial twohybrid system reporter strain, and didn＇t have the property of self-activation. 【Conclusions】Bacterial twohybrid pBT/ie1-exon4 bait plasmid without self-activation property was successfully constructed, and it can be used to screen library.