堆形艾美耳球虫早熟株生物学特性及其抑制消减cDNA文库的构建
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摘要
鸡球虫病是由艾美耳球虫寄生于鸡肠道所引起的一种危害严重的全球性寄生虫病,给养鸡业造成巨大的经济损失。目前控制球虫病的主要方法包括化学药物防治和疫苗免疫预防,但由于这些方法存在安全、价格以及抗药虫株出现等问题,迫切需要寻找新的防治手段来控制球虫病。研制高效安全抗球虫病基因工程疫苗的关键在于寻找到重要的虫体抗原基因。
本研究为了研究堆形艾美耳球虫(Eimeria. Acervulina)早熟株与母株之间基本生物学特性的差异,运用Jeffers创立的艾美耳属球虫早熟株选育方法对堆形艾美耳球虫(E. acervulina)进行了连续18代的早熟选育,并通过对早熟株与母株的孢子化卵囊大小、繁殖能力、致病性及免疫保护力等指标进行测定,分析了E. acervulina早熟株与母株之间基本生物学特性的差异。
为了进一步从分子水平对E. acervulina早熟株和母株之间的差异进行分析,本研究分别以早熟株和母株孢子化卵囊互为驱动组和实验组,利用抑制性消减杂交技术,进行了双向抑制性消减杂交,构建了2个抑制性消减cDNA文库。随机从2个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个消减cDNA文库的重组率为97%和98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从早熟株消减文库中获得了20个单一有效序列,其中9个单一序列与已知的蛋白同源性很高,主要为堆形艾美耳球虫丝氨酸蛋白酶抑制剂、表面抗原,刚地弓形虫假想蛋白、隐孢子虫假想蛋白、泰勒虫假想蛋白、弓形虫聚合酶等。从母株消减文库中获得了21个单一有效序列,有7个单一序列与已知的蛋白同源性很高,主要为刚地弓形虫假想蛋白、疟原虫假想蛋白、人类ATP合成酶、鼠类线粒体醛脱氢酶前体等。
根据序列分析获得的ESTs,从每个文库中选择4个阳性克隆基因,利用Real-time PCR进行分析。结果显示从母株消减cDNA文库中获得的ESTs,在母株孢子化卵囊中mRNA转录水平高于早熟株孢子化卵囊,而从早熟株消减cDNA文库中获得的ESTs,在早熟株孢子化卵囊中mRNA转录高于母株,这进一步说明利用SSH技术构建的E.acervulina早熟株和母株孢子化卵囊双向抑制性消减cDNA文库是比较好的,可用于进一步筛选早熟株与母株差异表达基因。
Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry. At present, conventional disease control strategies have relied on prophylactic medication and immunization with live vaccines. Due to the emergence of drug resistant parasite and the danger of live vaccines etc, Novel approaches are urgently needed to study to control coccidiosis. At present, identification important parasite antigen genes are crucial for the design of novel control approaches.
To find differences of characteristics between precocious and parent strains of E. acervulina, 18 passages were selected by serial through chickens apply to a method as described by Jeffers. Analyzed differences between the two strains according to the sizes of sporulated oocysts, reproduction and pathogenicity.
In order to analysis and identify differentially expressed genes from precocious and parent strains of E. acervulina, sporulated oocysts of two strains were used as the driver and the tester, respectively. Two subtractive cDNA libraries were constructed using the suppression subtractive hybridization (SSH) technique. PCR amplification revealed that the two subtractive cDNA libraries of precocious strains and parent strains contained approximately 97% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from each of two subtractive cDNA libraries. twenty unique sequences(ESTs) were found from the subtractive cDNA library of precocious strains of E.acervulina, nine ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Cryptosporidium muris, Eimeria acervulina serpin, Eimeria acervulina merozoite surface antigen, poly(A) polymerase of Toxoplasma gondii, etc. Twenty one unique sequences(ESTs) were found from the subtractive cDNA library of parent strains of E.acervulina, seven ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Plasmodium falciparum, Protein F and A of Enterobacteria phage, eukaryotic translation initiation factor 4 of Homo sapiens, and so on. Four differentially expressed genes obtained from two subtractive cDNA libraries conformed by Real-time PCR, respectively, which demonstrated that these genes were indeed differentially expressed. These results have provided the foundation for selecting differentially expressed genes of precocious and parent strains of E.acervulina and further studying new approaches to control coccidiosis.
本研究为了研究堆形艾美耳球虫(Eimeria. Acervulina)早熟株与母株之间基本生物学特性的差异,运用Jeffers创立的艾美耳属球虫早熟株选育方法对堆形艾美耳球虫(E. acervulina)进行了连续18代的早熟选育,并通过对早熟株与母株的孢子化卵囊大小、繁殖能力、致病性及免疫保护力等指标进行测定,分析了E. acervulina早熟株与母株之间基本生物学特性的差异。
为了进一步从分子水平对E. acervulina早熟株和母株之间的差异进行分析,本研究分别以早熟株和母株孢子化卵囊互为驱动组和实验组,利用抑制性消减杂交技术,进行了双向抑制性消减杂交,构建了2个抑制性消减cDNA文库。随机从2个cDNA消减文库中分别挑取50个克隆,经PCR鉴定2个消减cDNA文库的重组率为97%和98%。从每个文库中随机挑取50个克隆测序,并进行同源性比较分析,结果显示:从早熟株消减文库中获得了20个单一有效序列,其中9个单一序列与已知的蛋白同源性很高,主要为堆形艾美耳球虫丝氨酸蛋白酶抑制剂、表面抗原,刚地弓形虫假想蛋白、隐孢子虫假想蛋白、泰勒虫假想蛋白、弓形虫聚合酶等。从母株消减文库中获得了21个单一有效序列,有7个单一序列与已知的蛋白同源性很高,主要为刚地弓形虫假想蛋白、疟原虫假想蛋白、人类ATP合成酶、鼠类线粒体醛脱氢酶前体等。
根据序列分析获得的ESTs,从每个文库中选择4个阳性克隆基因,利用Real-time PCR进行分析。结果显示从母株消减cDNA文库中获得的ESTs,在母株孢子化卵囊中mRNA转录水平高于早熟株孢子化卵囊,而从早熟株消减cDNA文库中获得的ESTs,在早熟株孢子化卵囊中mRNA转录高于母株,这进一步说明利用SSH技术构建的E.acervulina早熟株和母株孢子化卵囊双向抑制性消减cDNA文库是比较好的,可用于进一步筛选早熟株与母株差异表达基因。
Avian coccidiosis is the major parasitic disease of poultry infected by Eimeria spp. It inflicts severe economic losses on the poultry industry. At present, conventional disease control strategies have relied on prophylactic medication and immunization with live vaccines. Due to the emergence of drug resistant parasite and the danger of live vaccines etc, Novel approaches are urgently needed to study to control coccidiosis. At present, identification important parasite antigen genes are crucial for the design of novel control approaches.
To find differences of characteristics between precocious and parent strains of E. acervulina, 18 passages were selected by serial through chickens apply to a method as described by Jeffers. Analyzed differences between the two strains according to the sizes of sporulated oocysts, reproduction and pathogenicity.
In order to analysis and identify differentially expressed genes from precocious and parent strains of E. acervulina, sporulated oocysts of two strains were used as the driver and the tester, respectively. Two subtractive cDNA libraries were constructed using the suppression subtractive hybridization (SSH) technique. PCR amplification revealed that the two subtractive cDNA libraries of precocious strains and parent strains contained approximately 97% and 98% recombinant clones, respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from each of two subtractive cDNA libraries. twenty unique sequences(ESTs) were found from the subtractive cDNA library of precocious strains of E.acervulina, nine ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Cryptosporidium muris, Eimeria acervulina serpin, Eimeria acervulina merozoite surface antigen, poly(A) polymerase of Toxoplasma gondii, etc. Twenty one unique sequences(ESTs) were found from the subtractive cDNA library of parent strains of E.acervulina, seven ESTs shared significant identity with the former, including hypothetical protein of Toxoplasma gondii, hypothetical protein of Plasmodium falciparum, Protein F and A of Enterobacteria phage, eukaryotic translation initiation factor 4 of Homo sapiens, and so on. Four differentially expressed genes obtained from two subtractive cDNA libraries conformed by Real-time PCR, respectively, which demonstrated that these genes were indeed differentially expressed. These results have provided the foundation for selecting differentially expressed genes of precocious and parent strains of E.acervulina and further studying new approaches to control coccidiosis.
引文
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