Screenning The Human Cell Proteins That Interact With CVB3VP3Protein

Objective:To screen the human cell proteins from the cDNA library of human heart, thatinteract with CVB3VP3protein.Methods:1. The VP3gene was amplified by PCR. PCR product of VP3was inserted intovector pGBKT7, then the recombinant bait plasmid pGBKT7-VP3was obtained.2. Using the LiAc-mediated method, the plasmid pGBKT7-VP3wastransformed into yeast strain AH109, then phenotype identification and PCR wereapplied to confirm whether the plasmid pGBKT7-VP3was transformed into yeaststrain AH109.3. Expression of VP3protein yeast strain AH109was detected by Westernblotting.4. VP3protein for auto-transcriptional activation of reporter genes (HIS3,ADE2and MEL1) was tested by substrate of enzyme X-α-Gal and nutrition-deficientmedium.5. The mating efficiency of VP3protein was tested by Small-scale Yeast Mating.6. Large-scale Yeast Mating was applied to screen the cDNA library of humanheart with CVB3VP3.7. Phenotype identification, PCR amplification and Alu I cutting were applied tosort positive colonies, and the positive colonies were sequenced and homologicallyanalyzed;8. The interaction activities between CVB3VP3and positive proteins weretested using α-galactosidase assay.9. The effects of the positive proteins on auto-transcriptional activation of thereporter genes (HIS3, ADE2and MEL1) were tested by the substrate of enzyme X-α-Gal and nutrition-deficient medium.10. The interaction between VP3and positive proteins were confirmed twice by Re-hybrid.Result:1. The sequencing confirmed that VP3gene was inserted into the multi-cloningsite of vector pGBKT7successfully. It also suggested that the open reading frame ofVP3gene in the recombinant bait plasmid pGBKT7-VP3was the same as that ofVP3gene in CVB3.2. Phenotype identification and PCR verified that plasmid pGBKT7-VP3wastransformed into the yeast strain AH109.3. Western blotting confirmed that VP3protein was expressed in yeast strainAH109.4. X-α-Gal assay and Nutrition-deficient medium suggested that VP3proteincould not auto-transcriptionally activate the reporter genes (HIS3, ADE2and MEL1).5. Small-scale Yeast Mating suggested that the expressing of VP3protein inyeast strain AH109could not affect the mating of yeast strains.6. We obtained50candidate clones in the cDNA library of human heart byLarge-scale Yeast Mating.7.50candidates were sorted by10types; Sequencing and homologicalalignment confirmed10following proteins: aldehyde dehydrogenase2family(mitochondrial)（ALDH2）, endoplasmic reticulum-golgi intermediate compartment(ERGIC)1（ERGIC1）, eukaryotic translation initiation factor4A2（EIF4A2）,troponin I type3(cardiac)（TNNI3）, leiomodin3(fetal)(LMOD3), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctionalprotein), beta subunit (HADHB), asparaginase homolog (S. cerevisiae)(ASPG),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), creatine kinase, muscle(CKM) and actin, alpha1, skeletal muscle (ACTA1)；8. α-galactosidase assay verified that positive proteins strongly interacted withVP3protein respectively.9. It was confirmed that the10positive proteins could not auto-transcriptionallyactivate the reporter genes (HIS3, ADE2and MEL1).10. Using re-hybrid experiment, we double confirmed the interaction between VP3and positive proteins.Conclusions:1. The recombinant plasmid pGBKT7-VP3has been constructed successfully.2. CVB3VP3protein fits for Yeast Two-hybrid System as a bait protein.3. This study successfully screened10positive proteins from the cDNA Libraryof human heart using bait protein CVB3VP3: ALDH2, ERGIC1, EIF4A2, TNNI3,LMOD3, HADHB, ASPG, GAPDH, CKM and ACTA1.