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目的构建和鉴定人巨细胞病毒(HCMV)UL123基因外显子2,3(ie1-exon2,3)的诱饵质粒,并评价其用于筛选胎儿脑文库的可行性。方法以重组质粒pTW IN1/ie1为模板扩增ie1-exon2,3,克隆入pBT质粒,转化E.coli XL1-B lue MRF’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W estern B lot对表达产物进行鉴定,并进一步鉴定重组子自身激活作用。结果在细菌双杂交系统中成功构建了细菌双杂交诱饵质粒pBT/ie1-exon2,3,并在报告菌株中表达了重组融合蛋白rIE1-N85/λC1,且pBT/ie1-exon2,3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-exon2,3,可用于筛选胎儿脑文库。
Objective To construct and identify bait plasmid of human cytomegalovirus (HCMV) UL123 gene exon 2 and 3 (ie1-exon2,3) and evaluate its feasibility for screening fetal brain library. Methods The recombinant plasmid pTW IN1 / ie1 was used as a template to amplify ie1-exon2,3 and cloned into pBT plasmid. The recombinant plasmid was transformed into E. coli XL1-B lue MRF’Kan. The positive recombinant was identified by restriction enzyme digestion and sequencing. The positive recombinant plasmids were transformed into the bacterial two-hybrid system to report the strain, the recombinant fusion protein was induced to express, and the expression products were identified by SDS-PAGE and Western blot, and the recombinant self-activation was further identified. Results The two-hybrid bacterial bait plasmid pBT / ie1-exon2,3 was successfully constructed in bacterial two-hybrid system and the recombinant fusion protein rIE1-N85 / λC1 was expressed in the reported strain, and pBT / ie1-exon2,3 did not activate effect. Conclusion The bait plasmid pBT / ie1-exon2,3 without self-activation was successfully constructed and could be used to screen fetal brain library.