本文選題：人呼吸道合胞病毒 + 裂解��； 參考：《中國協(xié)和醫(yī)科大學》2009年碩士論文

【摘要】： 目的采用成熟的病毒裂解純化疫苗制備技術(shù),以人類呼吸道合胞病毒(humanrespiratory syncytial virus,HRSV)野毒株中能誘導保護性抗體的F蛋白和G蛋白為目標蛋白,研究有效裂解HRSV和純化目標蛋白的方案。為獲得免疫原性強、安全性高的HRSV裂解純化亞單位疫苗做前期基礎研究。方法本研究分為兩部分,一部分是通過SDS-PAGE蛋白電泳觀察Triton X-100、NP-40等幾種常見裂解劑,在時間、溫度和濃度三種因素改變的情況下對HRSV裂解的效果,以篩選出針對目標蛋白裂解效果較好的裂解方案,并對這些裂解方案裂解過的樣品進行蛋白免疫印跡(Western-blot)實驗和透射電鏡檢查。另一部分是以蔗糖密度梯度離心法對裂解后的樣品進行目標蛋白的純化。摸索這種方法下,針對目標蛋白較佳的純化條件。繼而純化出一定量的目標蛋白并免疫小鼠,一月后處死采血,再進行中和實驗驗證我們純化出的目標蛋白的免疫原性和檢測其抗體效價。結(jié)果4%的去氧膽酸鈉37℃作用樣品2h、2%的NP-40 37℃作用樣品2h及1%Triton X-100作用90min對HRSV的裂解效果較好,其中前兩者均可在電泳圖上找到63KD和32KD的目標蛋白,且條帶清晰。1%Triton X-100 4℃作用90min的HRSV樣品雖然未能在電泳圖上觀察到32KD的目標蛋白,但63KD的目標條帶清晰粗大,有利于此目標條帶的純化。電鏡結(jié)果顯示它們均對HRSV進行了有效的裂解。Western-blot實驗均可識別此三種裂解方案裂解后的樣品63KD處的蛋白條帶。在對兩個目標蛋白的純化條件的摸索中,我們發(fā)現(xiàn)應用梯度為10%、15%、20%、25%和50%的不連續(xù)蔗糖密度梯度離心法,4℃、40000rpm離心16h對63KD的目標蛋白純化效果好,三種裂解方案裂解后的63KD的目標蛋白在15%和20%兩個蔗糖梯度層均有層積,后續(xù)動物實驗及中和實驗提示,在20%處的63KD的目標蛋白具有一定的免疫原性,抗體效價的平均值為1∶4。結(jié)論實驗中,篩選出三種較理想的裂解方案,并純化出一定量63KD的目標蛋白作為實驗性疫苗,動物實驗結(jié)果顯示,這種方法純化的實驗性疫苗可誘導機體產(chǎn)生機體免疫應答,中和抗體效價為1∶4-1∶8左右。為進一步研制HRSV亞單位疫苗積累了一定的原始資料。
[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，

本文編號：1814510