【摘要】 【目的】构建吕氏泰勒虫裂殖子cDNA表达文库,并从中筛选抗原候选基因。【方法】从吕氏泰勒虫裂殖子直接提取和纯化mRNA,采用oligo(dT)引物合成双链cDNA,并在其两端加EcoRⅠ/HindⅢ定向接头。将所产生的cDNA分子定向克隆到具有EcoRⅠ/HindⅢ粘性末端的λSCREEN载体的两臂之间。用PhageMaker extract对连接产物进行体外包装以形成完整的噬菌体,并用之转染大肠杆菌ER1647,从而构建成吕氏泰勒虫的cDNA表达文库。用吕氏泰勒虫阳性血清和兔抗绵羊IgG-AP筛选得到阳性克隆,经测序和Blast软件分析并获得新基因。【结果】成功构建吕氏泰勒虫裂殖子cDNA表达文库,其初级库容量约为1.0×106PFU,扩增文库的滴度为8.2×108PFU·ml-1,文库重组率为100%;通过免疫学筛选、测序和Blast软件分析,共获得30个新基因,其中15个基因已登录GenBank/NCBI。【结论】为研究泰勒虫疫苗、新型医药和诊断抗原,以及发展可持续性防制羊泰勒虫病提供基本材料。更多还原

【Abstract】 【objective】 A cDNA expression library of the merozoites of Theileria luwenshuni was successfully constructed and antigenic genes were obtained by immunoscreening from it.【Method】 Purified mRNA was isolated from the merozoites of Theileria luwenshuni.A library of oligo(dT)-primed cDNA with added directional EcoRⅠ/HindⅢ linkers was constructed and ligated to the EcoR I/Hind III arms of the screen vector.The recombinant phage DNA was packaged by using PhageMaker packaging extracts,resulting in a primary cDNA library.The positive clones were immunoscreened with the positive sera against Theileria luwenshuni and the rabbit anti-sheep IgG-AP from the library.The new genes were obtained by Blast anlysis and sequencing.【Result】 A cDNA expression library of the merozoites of Theileria luwenshuni was successfully constructed,with a size of 1.0×106 PFU,amplified cDNA library with a phage titer of 8.2×108 PFU.ml-1,and a recombinant rate of 100%.Thirty new genes were obtained by analysis of immunoscreening,sequencing and Blast,and 15 of them have been submitted in GenBank/NCBI.【Conclusion】The experiment has established an important material bases for the development of Theileria vaccine,new pattern medicine,diagnostic antigen and so on.更多还原