Cloning and expression of human cytomegalovirus UL123 gene exon 2,3 in bacterial two-hybrid bait plasmid / 中国医师杂志

ABSTRACT

Objective To clone and express HCMV UL123 gene exon 2,3(ie1-exon2,3) in bacterial two-hybrid bait plasmid,and identify its self-activation property.Methods HCMV ie1-exon2,3 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.The positive recombinant was identified by PCR,restriction enzyme digestions and sequencing analysis.The verified plasmid was transformed into bacterial two-hybrid system reporter strain.The soluble fusion protein was analyzed by SDS-PAGE and Western blot.The self activation effect of the recombinant was then tested.Results Bacterial two-hybrid pBT/ie1-exon2,3 bait plasmid was successfully constructed.The corresponding soluble fusion protein rIE1-N_(85)/?C1 was expressed in bacterial two-hybrid system reporter strain,and didn't show self-activation property.Conclusion Bacterial two-hybrid pBT/ie1-exon2,3 bait plasmid without self-activation property was successfully constructed,and it can be used to screen the library of fetal brains.