吐温80对戊型肝炎病毒病毒样颗粒稳定性的研究

Effect of Tween 80 on the stability of hepatitis E viral-like particles

目的探讨不同浓度吐温80对戊型肝炎病毒(hepatitis E virus)病毒样颗粒(virus-like particle,VLP)稳定性的影响以及对比吐温80与海藻糖及山梨醇对VLP的稳定性作用,为VLP疫苗制剂优化提供一定的参考。方法验证4种不同浓度(0.0025%、0.0050%、0.0100%和0.0200%)的吐温80对戊型肝炎病毒VLP稳定性的作用,在-80℃将戊型肝炎病毒VLP反复冻融3次,检测不同试验组VLP粒径大小、蛋白纯度及相对含量。在筛选出适宜浓度的吐温80后,选择15.0000%海藻糖及10.0000%山梨醇,分别对比3种不同类型的保护剂对戊型肝炎病毒VLP的保护作用。结果在不同浓度吐温80冻融试验中,对照组(冻融后)VLP平均粒径大小均远超出质量标准,而4种浓度的吐温80组VLP粒径大小均在质量标准范围内。经过方差分析,对照组(冻融前)与试验组各组峰面积差异无统计学意义(P=0.0531,P>0.05),对照组(冻融后)与试验组各组峰面积之间差异有统计学意义(P=0.0053,P<0.05)。同时,随着吐温80浓度的增加,VLP蛋白相对含量也逐渐增加。在不同类型保护剂冻融试验中,海藻糖组、山梨醇组以及对照组(冻融后)VLP平均粒径大小均不符合质量标准,仅0.0100%吐温80组3批次VLP平均粒径为56.27 nm,处于质量标准内。同时,体积排阻色谱检测结果显示,山梨醇组VLP蛋白纯度低于95.00%,山梨醇组和海藻糖组VLP蛋白纯度低于0.0100%吐温80组。经过t检验分析,与对照组(冻融前)相比,对照组(冻融后)以及3组试验组峰面积均出现下降,其中对照组(冻融后)下降幅度最大,10.0000%山梨醇组峰面积下降幅度次之,且峰面积差异具有统计学意义(P=0.0007,P<0.01),而15.0000%海藻糖组和0.0100%吐温80组峰面积差异无统计学意义(P=0.0723,P=0.2569,P>0.05)。另外,电镜下可以观察到在0.0100%吐温80组冻融后大部分VLP形态均一,未聚集成团。结论0.0100%吐温80对戊型肝炎病毒VLP具有良好的稳定作用。

Objective To investigate the effect of different concentrations of Tween 80 on the stability of virus-like particle(VLP)of hepatitis E virus,and to compare the stability of Tween 80 with that of trehalose and sorbitol on the stability of VLPs of hepatitis E virus,in order to provide a certain reference for optimizing of vaccine preparation.Methods To verify the stability of VLPs of hepatitis E virus with four different concentrations of Tween 80(0.0025%,0.0050%,0.0100%,and 0.0200%),VLPs was freeze-thawed repeatedly for three times at-80℃,and VLP particle size,protein purity,and relative content were detected,and the concentration of Tween 80 was determined after evaluation.Simultaneously,15.0000%trehalose and 10.0000%sorbitol were selected to compare the protective effects with that of Tween 80.Results In the freeze-thaw test with different concentrations of Tween 80,the average particle size of VLP in the control group(without adding Tween 80)was far exceeded the quality standard after freeze-thaw,while the particle sizes of VLPs in the four concentrations of Tween 80 groups were still within the quality standard.There was no significant difference in peak area between the control group(before freeze-thaw)and the experimental group(P=0.0531,P>0.05),while statistical differences laid between the control group(after freeze-thaw)and the experimental group(P=0.0053,P<0.05).Meanwhile,as the concentration of Tween 80 increased,the relative content of VLP protein also gradually increased.In the freeze-thaw test of different types of protective agents,the average particle size of VLPs in the trehalose group,sorbitol group and control group did not meet the quality standard.While in 0.0100%Tween 80 group,the average particle size of 3 batches of VLP was 56.27 nm,within the quality standard.Furthermore,the purity of VLPs in the sorbitol group was lower than 95%(quality standards),and VLP purity in 0.0100%Tween 80 group was higher than that in both trehalos and sorbitol group compared with the control group(before freeze-thaw),the peak area of the control group(after freeze-thaw)and the three experimental groups decreased significantly,the control group(after freeze-thaw)had the largest decrease,followed by the 10.0000%sorbitol group(P=0.0007,P<0.01),while 15.0000%trehalose group and 0.0100%Tween 80 group showed no difference(P=0.0723,P=0.2569,P>0.05)between them.In addition,under the electron microscope,most of the VLPs in the Tween 80 group were homogeneous after freeze-thaw,and did not aggregate into clusters.Conclusion Tween 80 has a good stabilizing effect on VLP of hepatitis E virus.