Preliminary Study On The Function Of Chloride Intracellular Channel Protein

Objective To investigate proteins associated with chloride intracellular channel 1 (CLICl) by yeast two-hybrid technique and, thus to clarify the functions of CLICl. To construct and identify the recombinant expression vector pCDGFP-CLIC2B.Methods The cDNA fragment of CLIC1 was amplified from pACT2-CLIC1 plasmid by PCR, and inserted into the bait vector pGBKT7 in frame. The recombinant pGBKT7-CLIC1 was then transformed into yeast host strain AH109, followed with transformation of human Hela cell cDNA library. Candidate positive clones were screened by SD/-Leu/-Trp/X-a-gal. The plasmids isolated from candidate positive clones was transformed E.coli using electroporation. The plasmids extracted from bacteria were sequenced, and then retrieved with Blast program. The plasmids with the known cDNA sequence (registered in GenBank) were cotransformed with pGBKT7-CLIC1 into AH109 to confirm the proteins interaction.In addition, PCR method was used to amplify the full length cDNA fragment of CLIC2B from pACT2-CLIC2B plasmid, and the product was ligated into the T vector to get the recombinant plasmid pUCm-T-CLIC2B. The recombinant plasmid was digested by the restriction enzyme, and the gene CLIC2B sequence was inserted into pCDGFP to contruct expression vector pCDGFP-CLIC2B, which was confirmed by DNA sequencing.Results The recombinant plasmid pGBKT7-CLIC1 was successfully constructed and that CLIC1 doesn't autonomously activate reporter genes. Sixteen positive clones which were screened by yeast two-hybrid system were sequenced, and four of them were...