Construction Of Yeast Two-Hybrid System And Screening And Identificaton Of Interactive Proteins Of CLN8P

Objective: The neuronal ceroid lipofuscinoses (NCLs), a group of progressive neurodegenerative disorders in human, are characterized by intralysosomal accumulations of ceroid lipopigment material in the form of granular, curvilinear, fingerprint, rectilinear and mixed profiles. The NCLs comprise at least ten recessive diseases (NCL1- NCL10) and CLN8 is the gene that induces NCL8. The function of CLN8 and its role in the NCLs pathogenesis is not clear now. Our aim is to screen and identify the interactive protein of CLN8P, provide clues for the studies on CLN8 function and pathogenesis of NCLs.Methods: 1) Interaction between the SV40 large T antigen and the P53 protein as a positive control was established for the yeast two -hybrid system. 2) CLN8 coding sequence was inserted into the BD vector (pLexA) in the proper orientation by gene engineering. And the constructed plasmid pLexA-CLN8 was introduced into host stains EGY48 (p8op-LacZ). Then the toxicity and self-activation of CLN8 protein on host strains were tested; 3) The Human Fetal Brain MATCHMAKER cDNA library (the "prey") was screened by yeast transformation, and Leu+ and LacZ+ positive yeast clones were obtained. 4) The plasmids were extracted from the obtained positive yeast clones, and inserted fragments detected by analysis of restriction enzyme digesting. Then only the plasmids with inserts were co-transformed to the EGY48 (p8op-LacZ) along with pLexA-CLN8 plasmid one to one by yeast simultaneous co-transformation so as to identify the protein interactions. 5) The inserted fragments of identified positive clones were sequenced and analyzed with blast in NCBI.Results: 1) The yeast two -hybrid system was established. 2) The bait plasmid pLexA-CLN8 was constructed and identified by restriction enzyme digesting and DNA sequencing. EGY48 (p8op-LacZ) -pLexA-CLN8 was established. CLN8P was...