人呼吸道合胞病毒裂解纯化疫苗的初步研究

发布时间：2018-04-28 08:21

  本文选题：人呼吸道合胞病毒 + 裂解　； 参考：《中国协和医科大学》2009年硕士论文

【摘要】： 目的采用成熟的病毒裂解纯化疫苗制备技术,以人类呼吸道合胞病毒(humanrespiratory syncytial virus,HRSV)野毒株中能诱导保护性抗体的F蛋白和G蛋白为目标蛋白,研究有效裂解HRSV和纯化目标蛋白的方案。为获得免疫原性强、安全性高的HRSV裂解纯化亚单位疫苗做前期基础研究。方法本研究分为两部分,一部分是通过SDS-PAGE蛋白电泳观察Triton X-100、NP-40等几种常见裂解剂,在时间、温度和浓度三种因素改变的情况下对HRSV裂解的效果,以筛选出针对目标蛋白裂解效果较好的裂解方案,并对这些裂解方案裂解过的样品进行蛋白免疫印迹(Western-blot)实验和透射电镜检查。另一部分是以蔗糖密度梯度离心法对裂解后的样品进行目标蛋白的纯化。摸索这种方法下,针对目标蛋白较佳的纯化条件。继而纯化出一定量的目标蛋白并免疫小鼠,一月后处死采血,再进行中和实验验证我们纯化出的目标蛋白的免疫原性和检测其抗体效价。结果4%的去氧胆酸钠37℃作用样品2h、2%的NP-40 37℃作用样品2h及1%Triton X-100作用90min对HRSV的裂解效果较好,其中前两者均可在电泳图上找到63KD和32KD的目标蛋白,且条带清晰。1%Triton X-100 4℃作用90min的HRSV样品虽然未能在电泳图上观察到32KD的目标蛋白,但63KD的目标条带清晰粗大,有利于此目标条带的纯化。电镜结果显示它们均对HRSV进行了有效的裂解。Western-blot实验均可识别此三种裂解方案裂解后的样品63KD处的蛋白条带。在对两个目标蛋白的纯化条件的摸索中,我们发现应用梯度为10%、15%、20%、25%和50%的不连续蔗糖密度梯度离心法,4℃、40000rpm离心16h对63KD的目标蛋白纯化效果好,三种裂解方案裂解后的63KD的目标蛋白在15%和20%两个蔗糖梯度层均有层积,后续动物实验及中和实验提示,在20%处的63KD的目标蛋白具有一定的免疫原性,抗体效价的平均值为1∶4。结论实验中,筛选出三种较理想的裂解方案,并纯化出一定量63KD的目标蛋白作为实验性疫苗,动物实验结果显示,这种方法纯化的实验性疫苗可诱导机体产生机体免疫应答,中和抗体效价为1∶4-1∶8左右。为进一步研制HRSV亚单位疫苗积累了一定的原始资料。
[Abstract]:Objective to prepare a mature purified vaccine against human respiratory syncytial virus (HRRSV), using F and G proteins, which can induce protective antibodies in human respiratory syncytial virus (HRRSV) wild strain, as the target proteins, and to study the scheme of effective cleavage of HRSV and purification of the target protein. In order to obtain HRSV cleavage purified subunit vaccine with strong immunogenicity and high safety, the preliminary basic research was done. Methods the study was divided into two parts. One was to observe the effect of Triton X-100 NP-40 and other common pyrolytic agents on HRSV cleavage by SDS-PAGE protein electrophoresis under three factors: time, temperature and concentration. In order to select a better cleavage scheme for the target protein, Western blot assay and transmission electron microscopy (TEM) were carried out on the samples which were cleavage from the target protein. The other part is the purification of the target protein by sucrose density gradient centrifugation. Under this method, the better purification conditions for the target protein were found. Then a certain amount of target protein was purified and the mice were immunized. After one month the blood was collected and the neutralization experiment was carried out to verify the immunogenicity of the purified target protein and to detect its antibody titer. Results 4% sodium deoxycholate at 37 鈩，

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