The Study Of IEX-1 Interaction Protein Using Yeast Two-hybrid System

OBJECTIVE:By screening of adult liver cDNA library use yeast two-hybrid to look for IEX-1 interaction protein and prepare for the further study of its functional mechanism.MATERIALS AND METHODS:Look for IEX-1 interaction protein through the screening of adult liver cDNA library using yeast two-hybrid technology. Extracted RNA from osteosarcoma MG-63 cells through Trizol method.RT-PCR amplified IEX-1 gene coding region, then through electrophoresis retrieved the amplified IEX-1 gene coding region and after SpeI/NotI double digestion they were connected with pDBLeu vector, thus expression plasmids were constructed. After detecting self-activation of bait proteins, screening Library and verification of rotary experiments we acquired true positive clones. Extracted the true positive fusion plasmid cDNA clones, sequenced and performed further analysis of biological information.RESUITS:1. During the Construction of fusion protein expression plasmid, we successfully acquired IEX-1 gene coding region fragments and constructed fusion protein expression plasmids pDBLeu-IEX-1,which We confirmed through restriction enzyme digestion and sequencing were cut out of the normal length inserts and vector backbone fragments.2. Transformed the recombinant pDBLeu-IEX-1 that was successfully constructed and empty vector pDBLeu into yeast AH109 to perform its toxicity and self-activation test. Then We found that there was no toxic effects for pDBLeu-IEX-1 to yeast AH109 and self-activation. 3. Used the bait plasmid pDBLeu-IEX-1 to transform competent cells, and cultured them at the temperature of 30℃for 3～8 days,then coated the bacteria on SD/-Trp/-Leu/-His/-Ade plate to select positive clones and screened the positive clones through X-a-gal color for further confirming. The results show that 12 positive clones were selected.4. Extracted the fusion plasmid of 12 cDNA clones, and transformed them together with pDBLeu-IEX-1 into yeast strain AH109 for rotary validation. Results showed that four clones of them were the true positive clones and the others were the false positive clones. Expanded and trained these four true positive clones and extracted plasmid for sequencing to identify the corresponding gene of the fusion plasmid cDNA. Finally four IEX-1 interaction proteins were determined:C reactive protein (CRP), Clusterin (CLU), Cathepsin B (CTSB), Heat shock protein 105 (HSPH1).CONCIUSION:1. Yeast two-hybrid system was successfully constructed and by the test it could be used for the further screening of IEX-1 interaction protein;2. Using yeast two-hybrid system We screened out four IEX-1 interaction protein:C reactive protein (CRP),Clusterin (CLU),Cathepsin B (CTSB),Heat shock protein 105 (HSPH1). Our experiment lays the foundation for the further pathogenesis study of osteosarcoma.