蜜蜂球囊菌cDNA文库的构建及胞外酶的定性研究
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摘要
蜜蜂白垩病(bee chalkbrood disease)是由蜜蜂球囊菌(Ascosphaera apis)引起的真菌性传染病,主要感染蜜蜂幼虫。白垩病是我国养蜂业中潜在病害之一,影响着我国养蜂业的发展。目前国内外对蜜蜂球囊菌的研究主要集中在病原的生物学特性、最适培养条件、发病流行规律、病理学、病情的诊断等方面,由于蜜蜂球囊菌的致病机理不明,不能在生产中有效的预防和治疗蜜蜂白垩病。我们采用Stratagene公司的ZAP Eepress(?)cDNA Synthesis Kit and ZAP Express(?)cDNA Gigapack(?)ⅢGold Cloning Kit试剂盒,构建了蜜蜂球囊菌cDNA文库。文库滴度测定为10~6 pfu·mL~(-1),扩增后的滴度是10~9 pfu·mL~(-1);噬菌体的转化后,蓝白斑筛选单菌落,重组率达到90%。这为后续工作的进行打下良好的基础。昆虫中肠围食膜主要由蛋白质、几丁质和糖类等组成。蜜蜂球囊菌通过分泌胞外蛋白酶、几丁质酶、酯酶和淀粉酶等破坏蜜蜂幼虫中肠围食膜侵染蜜蜂;本试验对几种常见的蛋白酶、酯酶和淀粉酶分别进行活性染色和银染,结果显示:蜜蜂球囊菌能够胞外分泌蛋白酶、酯酶和淀粉酶。在SDS-PAGE条件下,蛋白酶活性染色有两条条带,分子量为37.0 kDa和110.0 kDa;酯酶活性染色鉴定出三条带,分子量是14.1 kDa、20.1 kDa、29.0 kDa。在PAGE条件下,蛋白酶有两条条带,分子量为48.0 kDa和94.0 kDa;酯酶活性鉴定出一条带,分子量是35.0kDa淀粉酶活性鉴定出至少三条带,分子量为90.0 kDa、97.2 kDa和大于97.2 kDa。此外,我们还对球囊菌分泌几丁质酶的情况进行了探索。利用蜜蜂球囊菌胞外分泌物(几丁质结合蛋白)结合胶体几丁质(N-乙酰葡糖氨),设计不同结合时间、不同缓冲体系,进行结合蛋白试验,然后结合后进行Loading buffer和Calcofluor(M2R)处理;pH 7.4条件下结合6 d,Loading buffer处理的效果好,同时得出Loading buffer比Calcofluor(M2R)处理效果好。
Bee chalkbrood disease,caused by a pathological fungi,Ascosphaera apis mainly infect the honeybee brood.It is one of the main honey disease with great potential effect on apiculture development in China.The present research on chalkbrood are mainly on biological character of the Ascosphaera apis such as the optimal cultivating conditions,prevalence situation,pathology and diagnosis etc.Their pathogenic mechanism is still not clear today.How to control the disease is under exploration.A cDNA library was constructed using ZAP Express(?)cDNA Synthesis Kit and ZAP Express(?)cDNA Gigapack(?)ⅢGold Cloning Kit from Stratagene.The titre of the cDNA library was 10~6 pfu·mL~(-1),the titer of the amplification cDNA library was 10~9 pfu·mL~(-1),and the recombination rate is over 90%.Such a work will provide a platform for future ectracellular proteinase research.Peritrophic membrane is a thin layer membrane in midgut.It is consisted of protein,chitin and other carbonhydrate compounds.The fungi Ascosphaera apis infect the honeybee brood by secreting the extracellular enzyme such as proteinase,chitinase, esterase and amylase.In this study,we certificated that the extracellular enzyme including proteinase,esterase and amylase by activity staining and silver staining methods.There are two proteinase bands with molecular weight of 37.0 kDa and 110.0 kDa,three bands of esterase bands with molecular weight of 14.1 kDa,20.1 kDa,29.0 kDa under SDS-PAGE repectively.In the condition of PAGE,there are two proteinase bands with molecular weight of 48.0 kDa和94.0 kDa,one esterase bands with molecular weight of 35.0 kDa and three bands of amylase with with molecular weight of 90.0 kDa,97.2 kDa and more than 97.2 kDa;we also explore the chitin binding protein in the extracellular compositions using their chitin binding capability for different time under PBS buffer system.The binded protein were eluted by SDS-PAGE loading buffer and 1%Calcofluor(M2R)respectively.The results showed that the binding in PBS with pH 7.4 for 6 days and eluted with SDS-PAGE loading buffer was the best binding and elution parameter.
Bee chalkbrood disease,caused by a pathological fungi,Ascosphaera apis mainly infect the honeybee brood.It is one of the main honey disease with great potential effect on apiculture development in China.The present research on chalkbrood are mainly on biological character of the Ascosphaera apis such as the optimal cultivating conditions,prevalence situation,pathology and diagnosis etc.Their pathogenic mechanism is still not clear today.How to control the disease is under exploration.A cDNA library was constructed using ZAP Express(?)cDNA Synthesis Kit and ZAP Express(?)cDNA Gigapack(?)ⅢGold Cloning Kit from Stratagene.The titre of the cDNA library was 10~6 pfu·mL~(-1),the titer of the amplification cDNA library was 10~9 pfu·mL~(-1),and the recombination rate is over 90%.Such a work will provide a platform for future ectracellular proteinase research.Peritrophic membrane is a thin layer membrane in midgut.It is consisted of protein,chitin and other carbonhydrate compounds.The fungi Ascosphaera apis infect the honeybee brood by secreting the extracellular enzyme such as proteinase,chitinase, esterase and amylase.In this study,we certificated that the extracellular enzyme including proteinase,esterase and amylase by activity staining and silver staining methods.There are two proteinase bands with molecular weight of 37.0 kDa and 110.0 kDa,three bands of esterase bands with molecular weight of 14.1 kDa,20.1 kDa,29.0 kDa under SDS-PAGE repectively.In the condition of PAGE,there are two proteinase bands with molecular weight of 48.0 kDa和94.0 kDa,one esterase bands with molecular weight of 35.0 kDa and three bands of amylase with with molecular weight of 90.0 kDa,97.2 kDa and more than 97.2 kDa;we also explore the chitin binding protein in the extracellular compositions using their chitin binding capability for different time under PBS buffer system.The binded protein were eluted by SDS-PAGE loading buffer and 1%Calcofluor(M2R)respectively.The results showed that the binding in PBS with pH 7.4 for 6 days and eluted with SDS-PAGE loading buffer was the best binding and elution parameter.
引文
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