Research On Transcription Regulation Of Different IE180 Mutant On The Eukaryocyte Gene Expression

Immediate-early protein is the first viral protein synthesized after cell infected. In turn, they activate further viral promoters for the synthesis of early and late mRNA. IE protein have multiple functions, such as conversion of the host cell into a metabolically activate states, activation of viral early genes in the early phase, repression of their own transcription. IE 180 proteins can stimulate transcription from both homologous such as the ICP4 of HSV (herpes simplex virus) and heterologous viral and cellular promoter such as β-globe protein. It was devoted that class II promoter was stimulated by IE180.There is not TATA box on the upstream of the promoter, but there are binding sites of protein in the upstream of the promoter. David and his company identified IE gene in the human cytomegalovirus virus DNA segment. IE acts as a general gene transcription activator. It consists of DNA-binding domain and acid amino acid transcription activate domain. What kinds of promoter can be stimulated by IE 180? What kinds of promoter can be repressed byIE180? If IE180 can stimulate all early promote as described. As activator it can be applied to activate the foreign gene expression.From GenBank Accession No352564 we acquire a complete gene seqences. Anylysis the sequence about 1-1080 amino acid using the DNASIS tool .According to Esuro Ono and his company job. There is abundance of Ser in the 152-390 amino acid. Connecting with the sequence 'RRKRR' is a nuclei binding site. There is no characteristic in the amino acid sequence 63-152 and it is the piece that we want to delete to identify the function of the segment.IE180 gene mutants deleted the 64-151 amino acid was amplified by muti-PCR and were cloned by pMDIST-vector. The clone plasmids were named pJmP1P3P2.The segment corresponding to the sequence of 1-1079 amino acid of the GenBank sequence amplified by PCR, Its clone plasmids was named pJmP1P2.The segment corresponding to the sequence of 454-1079 of GenBank sequence amplified by PCR and its clone plasmids is named pJmP6P2-Three Clone plasmids and pcDNA3 were digested by restriction enzyme BamHI and Hind ,the gene segment of P1P2, P1P3P2, P6P2 were recycled. The 6.0kb segment was recycle. Conjected the 6.0kb segment and the segment of P1P2, P1P3P2, P6P2, three eukayotic expression recombinant plasmids, pcP1P2 pcP1P3P2 pcP6P2 were constructed .Cotransfected constructed plasimds with pCAT3-control into the HeLa cell andEmployed CAT (chloramphenical acetyl transferase) ELISA Assays to identified the function of constructed plasmid. The result indicates IE180 mutant P1P2 and P1P3P2 can transactivated the SV40 promoter. The mutant P6P2 repressed to SV40 promoter.Two outcoms can be devote: 1) The IE180 mutant deleted 64-151 amino acid still show as a transactivator to SV40 promoter. This result show the deleted fragment isn't the key domain to transactivation. 2) The mutant P6P2 translate a peptide includiing 165 amino acid and acted as inhibition to SV40 promote. It is show the mutant consists of a nuclei binding site and DNA-binding domain is enough to negative regulation.A report plasmid pCAT that we constructed has a CAT gene expressed by a CMV early promoter. Cotransfected pcP1P2 pcP1P3P2pcP6P2and pCAT into the HeLa cell. CAT (chloramphenical acetyl transferase) ELISA Assays to identified the function of constructed plasmid. The result indicates mutant P1P2, P1P3P2 and P6P2 repressed to CMV promoter. Same as IE promoter two special sequences 'ATCGT' located at two side of the transcription initial site of promoter. The IE 180 mutant combined with two special protein binding-sites and inhibited the promoter transcription.Accidentally in the approach showed that the plasimd pcDNA3 show as activator to SV40 and CMV early promoter. The result is acquired by instantaneous transinfect. The stability in the foreign gene expression needed advance identification.