Cell injury induced by 3D protein of different enterovirus 71 strains / 中华传染病杂志

ABSTRACT

Objective To compare the degree of cell injury induced by 3D protein (SDLY11 and SDLY107) of enterovirus 71 (EV71) strains.Methods EV71 strains SDLY11 and SDLY107 were respectively isolated from children with mild and severe hand foot mouth disease.The target genes 11-3D-Flag and 107-3D-Flag were amplified by reverse transcription-polymerase chain reation (RT-PCR) and inserted into the eukaryotic expression vector pcDNA3.1.The recombinant plasmids 11-3D-Flag-pcDNA3.1 and 107-3D-Flag-pcDNA3.1 were transformed into Escherichia.coli DH5α, respectively, and were identified by enzyme digestion and sequencing.The recombinant plasmids were transfected into rhabdomyosarcoma (RD) cells, respectively.Expression of 3D protein was detected by indirect immunofluorescence assay and western blot.Cell injury induced by 3D protein was detected with lactate dehydrogenase (LDH) test, cell proliferation was detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenylthiazolium bromide (MTT) test, and cell apoptosis was detected with Annexin-V and PI.Multiple comparisons among groups were analyzed using LSD-t test if multiple sets of variables were consistent with homogeneity of variance.If not, Dunnett T3 test was used.Results The 1 400 bp fragments were amplified by reverse tramscription (RT)-polymerase chain reaction (PCR), and the recombinant plasmids were digested by enzyme and the 1 400 bp and 5 400 bp fragments were obtained and identified.Gene sequencing showed that the sequences were consistent with the target genes.The specific fluorescence was observed by indirect immunofluorescence assay, and the western blot showed that the molecular weight of the target protein was 55×103.The LDH test showed that the A490 of SDLY11 3D protein transfection group (0.790±0.048) was higher than that of SDLY107 3D protein transfection group (0.641±0.018).The difference was statistically significant (t=5.14, P<0.05).The cell membrane damage caused by SDLY11 3D protein was more severe than SDLY107 3D protein.The MTT test showed that the A570 of SDLY11 3D protein transfection group (1.028±0.020) was lower than that of SDLY107 3D protein transfection group (1.081±0.002), and the difference was statistically significant (t=3.31, P<0.05).The effect on cell proliferation activity of SDLY11 3D protein was greater than SDLY107 3D protein.The results of Annexin-V/PI showed that the percentage of apoptotic cells of SDLY11 3D protein transfection group and SDLY107 3D protein transfection group were (1.471±0.246)% and (1.465±0.237)%, respectively, and the difference was not statistically significant (t=0.04, P=0.973).Conclusions Compared with the SDLY11 3D protein, SDLY107 3D protein induces slighter cell injury, has weaker effect on cell proliferation activity, and is more favorable for virus replication in cells.