Protein-protein Interaction Study Of P311

Hypertrophic scar is always formed after the healing of deep burn wound, however,the genes and the mechanism of regulation involved are not fully understood. We selected 97 genes in the study of the different expression genes of early hypertrophic scar and normal skin in the same burn patients, and found one gene,named p311(Genebank ID hsu36189),which was evidentially expressed in the scar. However, the role of p311 in the formation of fibrosis or scar remains unclear. Using fusion of GAL4 binding domain and P311 as the'bait'protein we screened the adult liver cDNA library and acquired the encoding gene of target protein. Herein is the major data.1. Test of the'bait'geneWe constructed the recombined plasmid pTZ57R/T-p311 after we got the kind gift of p311 which is sent by its founder Prof. Matthieu Levi-Strauss. Through PCR, then propagated, isolated and sequenced the plasmid in the positive bacteria clony which came from the blue-white plaque screening after pTZ57R/T-p311 was transformed into competent E. coli DH5α. The data demonstrated that the'bait'gene sequence (p311) was correct.2. Construction and transformation of the bait plasmidp311 cDNA was directly cloned into the multiclone sites Nde I,BamH I of pGBKT7 vector of GAL4 DNA-binding domain(DNA-BD) by recombinant technique.The recombinant of human p311 bait fusion gene, named pGBKT7-p311, was indentified by restriction endonuclease Nde I and BamH I, PCR and sequenced. Further more, pGBKT7-p311 was transformed into competent S.cerevisiae AH109 and screened the positive clony on the plate of SD/-Trp according to Yeast Transformation System 2 manual book (clontech). The positive clones were identified by PCR.3. Screen of the cDNA libraryBefore we sreened the cDNA library, we testified the toxicity effect and autonomous activation of pGBKT7-p311. The human adult liver cDNA library, which was fused with the GAL4-activation domain in the pACT2 vector, was pretranformed into Y187 obtained from Clontech. We gradually finished four-round screening of the library according to the...