肠道病毒71型不同病毒株3D蛋白对细胞损伤的作用

Cell injury induced by 3D protein of different enterovirus 71 strains

目的 比较肠道病毒71型（EV71）SDLY11株和SDLY107株3D蛋白对细胞的损伤程度.方法 EV71病毒株SDLY11和SDLY107分别取自轻症手足口病患儿和重症手足口病患儿.反转录PCR扩增目的基因11-3D-Flag和107-3D-Flag,分别将其插入真核表达载体pcDNA3.1中,连接产物转化至大肠杆菌DH5α,提取质粒进行酶切和测序鉴定.重组质粒转染人横纹肌肉瘤（RD）细胞,间接免疫荧光试验和蛋白质印迹检测3D蛋白表达,乳酸脱氢酶（LDH）检测3D蛋白导致的细胞损伤,噻唑蓝（MTT）比色法测定3D蛋白对细胞增殖的影响,Annexin-V/Pl双染法测定3D蛋白引起的细胞凋亡.各组间两两比较方差齐采用LSD-t检验,方差不齐采用Dunnett T3检验.结果 反转录PCR扩增获得1 400 bp片段,酶切鉴定重组质粒后分别获得1 400 bp和5 400 bp片段,测定序列与目的基因一致.间接免疫荧光试验观察到特异性荧光,蛋白质印迹显示目的蛋白相对分子质量为55×10^3.LDH结果显示SDLY11株3D蛋白转染组的吸光度（A）490值高于SDLY107株3D蛋白转染组,分别为0.790±0.048和0.641±0.018,差异有统计学意义（t=5.14,P〈0.05）,SDLY11株3D蛋白引起的细胞膜损伤情况较SDLY107株3D蛋白严重.MTT比色法结果显示SDLY11株3D蛋白转染组的A570值低于SDLY107株3D蛋白转染组,分别为1.028±0.020和1.081±0.002,差异有统计学意义（t=3.31,P〈0.05）,SDLY11株3D蛋白对细胞增殖活性的影响大于SDLY107株3D蛋白.Annexin-V/PI双染法结果显示,SDLY11株3D蛋白转染组和SDLY107株3D蛋白转染组的细胞凋亡百分比分别为（1.471±0.246）%和（1.465±0.237）%,差异无统计学意义（t=0.04,P=0.973）.结论 与SDLY11株3D蛋白相比,SDLY107株3D蛋白引起的细胞损伤程度较轻微,且对细胞增殖活性的影响较弱,更有利于病毒在细胞内的复制.

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.