The Screening Of The Proteins Interacting With HCMV Major Immediate-early Protein IE₂86

Objective: Proteins interacting with IE₂86 from the brain tissue of 12-week-old embryo were screened by Pull-Down assay, to investigate the role of IE₂86 in CNS anomalies induced by HCMV congenital infection and its possible mechanisms.Methods: (1) UL122 CDS encoding IE₂86 was amplified by PCR with pBT-IE₂86 plasmid as template, and then the amplification product was digested with restriction endonucleases BamHI and NotI, and cloned into the corresponding sites on bait plasmid pGEX-4T-1. The recombinant was transformed into E. coli BL21. Followed by PCR, restriction and DNA sequence analysis. (2) GST-IE₂86 fusion protein was expressed in 30℃for 3-4 hours under 1mM IPTG induction and purified by affinity column chromatography; GST protein was expressed and purified as negative control. (3) Fetal brain tissue from 12-week-old embryo was taking out and grinding into homogenate. The proteins interacting with IE₂86 were screened from lysates by Pull-Down assay. (4) Cut the stably expressive protein bands and analysed the proteins with Quatropde-Time of Flight Mass spectrometer.Results: (1) The pGEX-4T-1/IE₂86 bait plasmid was constructed successfully. The result of DNA sequence analysis was no mutation compared with the sequence in GenBank. (2) GST- IE₂86 fusion protein and GST protein were expressed and purified successfully. The protein molecular weight of GST- IE₂86 fusion protein was 92kDa, and GST protein was 26kDa, consistent with the expected protein molecular weight. (3) The fetal brain protein which involved with IE₂86 has been found by using the method of Pull-Down assay, after mass spectrometry detection, there are four kinds of protein: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin.Conclusions: HCMV IE₂86 may have interaction with four kinds of protein from 12-week-old embryo: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin, however, the biological significance and the role of these interaction in fetal congenital anomalies of the central nervous system (CNS) which caused by infection of HCMV need to be further studied.