| Objective: To screen CVB3 non-structure protein 3A interactive proteins in the human heart c DNA library by yeast two hybrid system; to analysis the interaction between 3A and the resulting cell protein TMED4 in order to study the molecular mechamnism of CVB3 infection.Methods: 1. Construction of recombinant bait vector of CVB3 3A: After the 3A gene was amplified by RT-PCR, the PCR product was inserted into the p GBKT7 and the bait plasmid p GBKT7-3A was obtained. 2. The recombinant bait plasmid p GBKT7-3A was transformed into yeast strain AH109 by the Li Ac-mediated method. And whether the plasmid p GBKT7-3A was transformed successfully or not was confirmed by phenotype identification and PCR method. 3. The expression of 3A protein in AH109 was detected by Western Blot. 4. SD/–Ade/–Trp and SD/–His/–Trp dropout supplement and X-α-Gal assay were used to select for anto-activation and toxicity of the bait plasmids, in addition, the mating efficiency of 3A was investigated by small-scale Yeast Mating. 5. To screen the human heart protein that interact with CVB3 3A from the c DNA library by Large-scale Yeast Mating. 6. Fifty-two positive colonies were sorted by Phenotype identification, PCR amplification and Alu I cutting. Moreover, homologically analyzed and sequenced were applied. 7. The positive proteins for activation of reporter genes(HIS3, ADE2 and MEL1) were tested by SD/–Ade/–Leu and SD/–His/–Leu dropout supplement and X-α-Gal assay, and the interaction between 3A and positive proteins were retested by yeast mating. 8. He La cells were collected 36 h post transfection by p Bud CE4.1-3A. 3A wasdisplayed by using Rhodanmine goat anti-mouse(red fluorescence) conbined with mouse anti-His tag antibody and TMED4 was displayed by using Alexa Flour 488 goat anti-rabbit conbined with rabbit anti-TMED4 antibody. Then the subcellular colocalization between 3A and TMED4 was observed by the high intension cell imaging analysis system. 9. He La cells were transfected by p Bud CE4.1-3A and the total cellular proteins were collected, then TMED4 was pulled down by anti-His tag in collected proteins. Finally, the expression of TMED4 was detected by Co-Immunoprecipitation. 10. He La cells were differently collected at 24 h, 36 h, 48 h post transfection by p Bud CE4.1-3A, and the CCK-8 method was used to detected the cell activity. 11. Annexin V method was used to investigate the apoptosis cells transfected by p Bud CE4.1-3A. 12. The effection of the plasmid p Bud CE4.1-3A on cell cycle: Cell cycle distribution was detected by flow cytometry after transfeced 24 h,36 h,48 h and 54 h. 13. The total cellular proteins of He La cells that transfected by p Bud CE4.1-3A at 0 h, 18 h, 24 h, 36 h, 40 h, 48 h, 54 h, 60 h and infected by CVB3 at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h were collected and detected by Western Blot.Result: 1. The result of sequencing confirmed that 3A gene was inserted into the multicloning site of p GBKT7 successfully, and the open reading frame of 3A gene in the recombinant plasmid p GBKT7-3A was identical with that of CVB3. 2. Phenotype identification and PCR verified that the recombinant plasmid p GBKT7-3A was transformed into the yeast strain AH109 successfully. 3. Western Blot also confirmed that the expression of fusion protein 3A in the yeast strain AH109. 4. SD/–Ade/–Trp and SD/–His/–Trp dropout supplement and X-α-Gal assay showed that 3A protein could not activate the reporter genes(HIS3, ADE2 and MEL1). Further, the expression of 3A in AH109 have no effect on mating efficiency between two mating yeast strains.5. Fifty-two candidate clones in human heart c DNA library were obtained by Large-scale Yeast Mating. 6. Six types proteins were got : Ryanodine Receptor 2(Ry R2), Signal peptidase complex subunit 2(SPCS2), Transmembrane emp24 protein transport domain containing 4(TMED4), Cytochrome c oxidase subunit I(COX1), Cytochrome c oxidase subunit III(COX3), Myosin heavy chain(MYH7) and Succinate dehydrogenase complex subunit D(SDHD). 7. It was confirmed that 6 positive proteins could not activated the reporter genes(HIS3, ADE2 and MEL1) and the interaction between 3A and positive proteins were verified preliminary by yeast mating. 8. 3A and TMED4 had the subcellular co-localization in the plasmid transfection cells by the high intension cell imaging analysis system. 9. 3A was coprecipitation with anti-TMED4 and TMED4 was coprecipitation with anti-His tag in the collectiong precipitate. The result showed that 3A and TMED4 could interact in the p Bud CE4.1-3A transfected cells. 10. The result of CCK-8 assay showed that transfection of recombinant plasmid p Bud CE4.1-3A can significantly inhibit the proliferation of He La cells at 36 h; 11. The result of Annexin V method showed that transfection of recombinant plasmid p Bud CE4.1-3A can significantly induce the apoptosis of He La cells at 36 h; 12. Flow cytometry analysis revealed: Compared with the mock transfected group which transfected with vector p Bud CE4.1, the proportion of G1 phase was increased at 54 h post transfection. 13. The expression of TMED4 in total protein transfected by p Bud CE4.1-3A and infected by CVB3 had obvious change trends: it was gradually reduced after transfection, and at the 36 h post transfection time the expression of TMED4 was least, but after 36 h, it had a trend to elevated; after infection the expression of TMED4 was gradually reduced and at the 9 h infection time became least, but after 9 h, it had a trend to elevated.Conclusions: 1. It was comfirmed that 3A could be a bait protein in yeast two-hybrid system, and we obtained 6 positive proteins from the human heart c DNA Library successfully: Ry R2 、 SPCS2 、 TMED4、 COX1、 COX3 and MYH7 /SDHD. 2. The interaction of 3A and host protein TMED4 was further confirmed. 3. The interaction between 3A and TMED4 take part in the cell apoptosis by regulated the expression of TMED4 in cells, and the cell activity was decreased under the interaction. |
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