柔嫩艾美耳球虫孢子发育阶段虫体抑制性消减文库的构建

Construction of Subtractive cDNA Libraries of the Sporogony Stage of Eimeria tenella by Suppression Subtractive Hybridization

摘要:

为了分离鉴定柔嫩艾美耳球虫（Eimeria tenella）孢子发育阶段虫体的差异表达基因，分别以柔嫩艾美耳球虫未孢子化卵囊和孢子化卵囊为驱动组、子孢子为实验组，或未孢子化卵囊为驱动组、孢子化卵囊为实验组，利用抑制性消减杂交（SSH）技术，构建了2个子孢子cDNA消减文库和1个孢子化卵囊cDNA消减文库。随机从3个cDNA消减文库中分别挑取50个克隆，经PCR鉴定2个子孢子cDNA消减文库的重组率都为96%，孢子化卵囊cDNA消减文库的重组率为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：从孢子化卵囊cDNA消减文库中获得了13个单一有效序列，其中8个EST与已知蛋白同源性很高；从2个子孢子cDNA消减文库中共获得了40个单一有效序列，其中9个EST与已知蛋白同源，其余可能为柔嫩艾美耳球虫的新基因。这些结果为分离柔嫩艾美耳球虫新功能基因和进一步探索防治球虫病的方法提供了理论基础。

Abstract:

In order to clone and identify differentially expressed genes in the sporogony stage of Eimeria tenella，the cDNAs from unsporulated oocysts and sporulated oocysts of E. tenella were used as driver，respectively，the cDNAs from sporozoites of E. tenella was used tester，Two subtractive cDNA libraries of sporozoites were constructed by using the technique of suppression subtractive hybridization (SSH). the cDNAs from unsporulated oocysts was used driver，the cDNAs from sporulated oocysts was used tester，one subtractive cDNA library of sporulated oocysts was constructed. PCR amplification revealed that the two subtractive cDNA libraries of sporozoites and one subtractive cDNA library of sporulated oocysts contained approximated 96%，96% and 98% recombinant clones，respectively. Fifty positive clones were sequenced and analyzed in GenBank with Blast search from three subtractive cDNA libraries，respectively，thirteen unique sequences were found from the subtractive cDNA library of sporulated oocysts，eight ESTs shared significant identity with previously described. A total of forty unique sequences were obtained from the two subtractive cDNA libraries，nine ESTs shared significant identity with previously described，the other sequences represent novel genes of E. tenella with no significant homology to the proteins in Genbank. These results have provided the foundation for cloning new genes of E. tenella and further studying new approaches to control coccidiosis.