Yeast Two-hybrid System Screening Halr Interacting Protein

The liver is a unique organ, and first in line, the hepatocytes encounter the potential to proliferate during cell mass loss. The control of hepatic growth and regeneration has interested experimentalists for much of the 20th century. Augmenter of liver regeneration (ALR), one of the latest hepatotrophic factors, is known to be live-specific. ALR specifically stimulates proliferation of cultured primary hepatocytes in vitro and enhances liver regeneration after liver partial hapatectomization in vivo. But it displays no significant effect on the proliferation of non-hepatocytes or tumor cell lines derived from tissues other than liver. Because of the target-specific activity, ALR was different from various well known non-specific hepatic stimulators such as insulin, EGF, TGF- α , IGF-I and HGF, which can stimulate proliferation of wide variety of cell types. However, the signaling mechanisms of ALR has been unclear. According to those, we seek for the genes encoding ALR-associated proteins from the human liver cDNA library using yeast two-hybrid system.Main procedures and methods:1. A hALR cDNA fragment was obtained by extracting mRNA from human fetal liver, and RT-PCR. It was inserted into DNA-BD vector pGBKT7 in the proper orientation to create a vector pGBKT7-hALR. The constructed plasmid was identified by restriction endonucleases digesting and DNA sequencing.2. The pGBKT7-hALR was introduced into yeast AH 109 by LiAc transformation method. The recombinant protein BD-hALR expression in the transformed yeast AH109(pGBKT7-hALR) was identified by SDS-PAGE and western blot. Nutritional selection screening and colony-lift filter assay of β -gal were used to test the BD-hALR protein for transcriptional activation function before using it to screen a library.3. AH109(pGBKT7-hALR) was mated with the pretransformed human liver cDNA library and the putative positive clones were obtained by nutritional selection screening and X- α -Gal assay.4. The total DNA was isolated from the putative positive clones with lyticase method, and then the AD plasmids were rescued via transformation of E.coli. To sort colonies, the AD library inserts were amplified by PCR and the PCR products were characterized by digesting with restriction enzyme. Yeast mating was performed to eliminate falase positives. Finally, the AD library inserts of the true two-hybrid positives were sequenced by using GAL4 AD sequencing primer and compared with those in...