Construction And Expression Of Eukaryotic Plasmid VP1Gene Of CVB3and Its Effection On HeLa Cells

Objective:To construct and identify the recombinant eukaryotic plasmid of CoxsackievirusB3VP1gene, and to detect its expression in HeLa cells. Then observe its effects oncell morphous, cell viability and cell cycle with the aim to investigate the molecularmechanism of CVB3infection.Methods:1. Construction of the plasmid pBudCE4.1-VP1: The VP1gene was amplifiedby PCR from a template of total mRNA of CVB3. The amplified DNA wasdirectionally inserted into vector pBudCE4.1. Then the recombinant plasmid wasidentified by restriction endonucleases digestion and sequencing;2. Expression of the plasmid pBudCE4.1-VP1: The expression of VP1genewas observed by Western blotting and immunofluorescently after transfection;3. The effection of the plasmid pBudCE4.1-VP1on HeLa cells was detected byMTT assay and observed the change of cell morphology by inverted microscope;4. To detect the interaction among the CVB3VP1protein and host proteinGM130by confocal microscopy with double immunofluorescent stain aftertransrected;5. The effection of the plasmid pBudCE4.1-VP1on cell cycle:Cell cycledistribution was detected by flow cytometry after transfeced16h,20h,24h,28h and32h.Result:1. The result of restriction endonucleases treatment and sequencing confirmthat VP1gene was successfully inserted into the vector pBudCE4.1in the samesequence to CVB3m type;2. The reusult of Western blotting and immunofluorescent show that the VP1gene expressed obviously in HeLa cells after transfected and the expression increasedover time; 3. After the VP1transfected into Hela cells12h, a typical cytopathetic effectwas observed in transfected HeLa cells; The reusult of MTT assay show thattransfection of recombinant plasmid pBudCE4.1-VP1can significantly inhibit theproliferation of HeLa cells;4. The reusult of double immunolabelled GM130and VP1showed that: withthe increased of VP1protein, the GM130was ribbon-like in normal cells and becomedisperse in the transfected cells;5. Flow cytometry analysis revealed: Compared with the mock transfectedgroup which transfected with vector pBudCE4.1, the proportion of G1phase wasincreased to58.81%(P＜0.05),57.15%(P＜0.05),54.87%(P＜0.01),55.46%(P＜0.05) and56.96%(P＜0.05) at16h,20h,24h,28h and32h post transfection.Conclusions:1. The eukaryotic expression plasmid VP1gene of CVB3was successfullyconstructed and express in HeLa cells;2. The VP1can cause significant cytopathic effect and inhibit the proliferationof HeLa cells;3. The golgi ribbon was brokendown and the structure of golgi apparatus wasdestroyed after transfection. VP1would be the key structure protein of causing Golgiapparatus destruction in the CVB3infected cells;4. After tansfecion with pBudCE4.1-VP1, the HeLa cell cycle should be mainlyarrest in G1phage.