论文摘要
鸡球虫病(Coccidiosis)是由一种或几种鸡艾美球虫引起的以肠道病变为主的寄生原虫病,对养禽业的危害很大,每年由鸡球虫病造成的直接和间接经济损失难以估计。巨型艾美球虫(Eimeria maxima)是诱发鸡球虫病的3种常见球虫之一,具有很强的免疫原性,对其它几种鸡球虫有部分的交叉免疫力,故在各种研制的鸡球虫病疫苗中,E.maxima是必不可少的虫种之一。但是,E.maxima株免疫原性差异最显著,在田间有时可因E.maxima免疫变异株的出现导致疫苗免疫失败。因此,疫苗的研制和使用有赖于对各地虫株的免疫原性进行深入研究,特别是免疫原性相异株间差异表达基因的研究。抑制消减杂交技术(suppression subtractive hybridization,SSH)是一种以抑制性PCR为基础的寻找差异表达基因的方法,是获得新基因乃至分子进化分析的重要研究手段。为了分离与鉴定E.maxima免疫原性相异株间的差异表达基因,探索球虫抗原变异的分子机理,为此,本研究首先以平均增重与病变记分减少率和卵囊减少率等为指标,对实验室保存的E.maxima南通株(NT)和E.maxima上海株(SH)进行了免疫原性差异的分析;然后,利用抑制消减杂交方法,分别以E.maxima SH株孢子囊cDNA为驱动组,以E.maxima NT株孢子囊cDNA为试验组,或以E.maxima NT株孢子囊的cDNA为驱动组,E.maxima SH孢子囊的cDNA为试验组,分别构建了2个抑制性消减文库,并对文库进行了分析。一、E. maxima南通株与上海株间的交叉保护力试验分别以平均增重与病变记分减少率和卵囊减少率等为指标,对实验室传代保存的E.maxima NT株和E.maxima SH株进行了株间交叉免疫力的分析。用E.maxima NT株和E.maxima SH株每鸡免疫接种2 500个、2周后每鸡攻击10×104个孢子化卵囊时,同源株攻击试验组鸡的平均增重与对照组相近(P> 0.05),病变记分减少率分别为79.55 %与88.46 %;E.maxima NT株免疫、E.maxima SH株攻击时,试验鸡的平均增重与对照组相近(P> 0.05),病变记分减少率为71.15 %;E.maxima SH株免疫、E.maxima NT株攻击时,试验鸡的平均增重与对照组间有显著差异(P< 0.05),病变记分减少率仅为36.36 %。用E.maxima NT株和SH株免疫接种100个、攻击100个孢子化卵囊时,同源株攻击后的卵囊减少率分别为100 %与97.56 %;E.maxima NT株免疫、E.maxima SH株攻击时的卵囊减少率为80.98 %;E.maxima SH株免疫、E.maxima NT株攻击时的卵囊减少率为41.20 %。结果显示:2株球虫对同源株攻击均能产生有效的保护力,E.maxima NT株对E.maxima SH株有交叉保护力,E.maxima SH株对E.maxima NT株无交叉保护力;这2株E.maxima的免疫原性特征没有随虫株在实验室的传代和保存而发生改变。二、E. maxima南通株与上海株间差异表达基因消减杂交文库的构建与初步分析为了分离与鉴定E.maxima SH株与E.maxima NT株间的差异表达基因,利用抑制消减杂交(SSH)方法,分别以E.maxima NT株孢子化卵囊cDNA为试验组(Tester),以E.maxima SH株孢子化卵囊cDNA为驱动组(Driver),或以E.maxima SH株孢子化卵囊cDNA作为Tester,E.maxima NT株孢子化卵囊cDNA作为Driver,通过两轮杂交和两次PCR后,将第二次PCR产物连接、克隆转化,经过蓝白斑筛选,分别构建了2个抑制性消减文库。随机从两个消减文库中分别挑取96个克隆,经PCR鉴定显示E.maxima SH株和E.maxima NT株孢子化卵囊cDNA消减文库的重组率分别为91 %和93 %,差异基因片段大小分布在0.25～1.0 kb之间。从每个文库中随机挑取30个阳性克隆测序,并进行同源性比较分析,结果显示:共获得了36个有效序列,其中E.maxima SH株的cDNA消减文库有4个单一序列与已知基因同源性较高,E.maxima NT株cDNA消减文库有3个单一序列与已知基因同源性较高,其余28个序列均无同源性相似基因,可能为E.maxima的新基因。这些结果将为分离E.maxima新的功能基因和进一步探索球虫抗原变异相关的分子机理奠定基础。
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