柔嫩艾美耳球虫裂殖子全长cDNA文库的构建及应用

为克隆柔嫩艾美耳球虫裂殖子阶段的功能基因的全长序列,利用SMART技术,采用Clontech公司的Creator TMSMARTTMcDNA Library Construction Kit构建了鸡球虫裂殖子的全长cDNA文库。用试剂盒提取mRNA后,以cDNA合成试剂盒合成cDNA。连接至pDNR-LIB载体,用电转化法将重组质粒转化到E.coli DH10B内得到原始文库,扩增后保存于-80℃冰箱内;最后以文库为模板,分别以研究室已成功克隆序列(EtSAG2、EtMIC-2、EtMCAT)及Sanger的部分EST序列(Contig1218)设计引物,进行PCR扩增并测序检验文库的实用性。经测定,构建的初级cDNA文库约含有8.72×107个重组子,插入的片段多在1kb～3kb之间,平均插入片段长度约1.8kb,扩增后文库保存的滴度为2.4×104 cfu/mL;通过文库的PCR扩增,不仅可以成功扩增出研究室已成功克隆的序列(EtSAG2、EtMIC-2),并成功获得EtMCAT和EST序列(Contig1218)的全长cDNA序列,经比对分析Contig1218所编码的蛋白为组织蛋白酶B样半胱氨酸蛋白酶(Cathepsin B)。结果表明,已成功构建了柔嫩艾美耳球虫裂殖子高质量的全长cDNA文库,从而为筛选鸡球虫的功能基因全长序列奠定了基础。

Construction of a cDNA Library for Merozoite of Eimeria tenella Using SMART Technique

A cDNA library for merozoite of Eimeria tenella was constructed by SMART technique using CreatorTM SMARTTM cDNA Library Construction Kit.The mRNA was extracted from merozoite of E.tenella using mRNA Isolation System.The cDNA was synthesized using cDNA Synthesis Kit.Then SMART cDNA was ligated to pDNR-LIB vector.The ligation mixture was transformed into E.coli DH10B by electroporation.Using the amplified library as template DNA,the primers were designed according to be successful cloned sequences in our research group(EtSAG2,EtMIC-2,EtMCAT) and the EST sequences from Sanger(Contig1218).The cDNA library contained 8.72×107 independent clones with DNA inserts of 1-3 kb,and the average length was 1.8 kb.And the genes known(EtSAG2,EtMIC-2,EtMCAT) and unknown(Contig1218) were amplified successful using this library as template DNA.So the results showed this library could serve as a valuable resource for screening and isolating functional genes for E.tenella.