摘要:
为制备高纯度的中华鳖虹彩病毒,分析其免疫相关抗原,采用3种方法纯化中华鳖虹彩病毒粒子并对其结构蛋白进行了初步分析。结果表明:差速离心法回收的样品中病毒纯度差、密度低,在电镜下不易观察到病毒粒子;病毒培养物经冻融、磁力搅拌、超声处理后,进行差速离心可提高病毒的回收率,但超声处理易造成病毒结构破坏;病毒培养物经离心浓缩、匀浆、磁力搅拌处理,再通过差速和蔗糖密度梯度二步离心纯化后,在蔗糖密度为30%～40%、40%～50%和50%～60%之间分层的样品中均可观察到病毒粒子,其中,蔗糖密度为50%～60%之间回收的病毒粒子纯度最高,且结构完好。SDS-PAGE分析表明,纯化病毒至少含20条蛋白条带,分子量为50kDa的核衣壳蛋白是中华鳖虹彩病毒的高丰度蛋白。Western-blot分析结果证实大多数蛋白条带能被鼠抗中华鳖虹彩病毒高免血清特异性地识别。
关键词:
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虹彩病毒 /
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中华鳖 /
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纯化 /
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分析
Abstract:
In order to obtain highly purified soft-shell turtle iridovirus(STIV) for immunological studies,3 methods were investigated.The purified virus was analyzed to certify its purity and protein structure.Results indicated an impurity and low recovery rate in the process applying two freezing-thawing cycles followed by differential centrifugation.Furthermore,the electron microscopic observation on the virus was difficult.On the other hand,by freezing-thawing the virus culture followed by magnetic stirring,ultrasonication and differential centrifugation,the virus recovery rate was improved.However,the virus structure was damaged.Alternatively,virus culture was concentrated by centrifugation,homogenization,magnetic stirring and differential centrifugation,in that order,and followed by sucrose density gradient centrifugation.The most satisfactory result was finally obtained.The virus particles collected could be observed in 30%-40%,40%-50% or 50%-60% sucrose density fractions.The most abundant and intact virions were seen in the 50%-60% fraction.The purified STIV virions were further subjected to SDS-PAGE.The result showed that the STIV contained more than 20 proteins,and that its major capsid protein corresponded to a 50kDa abundant protein.The Western-blot analysis further indicated that most of the protein bands could be specifically recognized by using high quality mouse anti-STIV serum.
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