莫氏巴贝斯虫裂殖子cDNA表达文库的构建及免疫学筛选

Construction and Immunoscreening of a Merozoites cDNA Expression Library of Babesia motasi

为了构建莫氏巴贝斯虫(Babesia motasi)裂殖子cDNA表达文库,从中筛选和鉴定功能基因,利用差速离心法从莫氏巴贝斯虫感染的红细胞中纯化裂殖子,提取总RNA并纯化mRNA。在合成的双链cDNA两端加上含EcoRⅠ和HindⅢ定向接头后连接到λSCREEN载体上。通过体外包装形成完整的噬菌体颗粒,并用之转染ER1647,构建莫氏巴贝斯虫裂殖子的cDNA表达文库。用莫氏巴贝斯虫阳性血清筛选cDNA文库,获得的阳性克隆,经过测序和Blast软件分析鉴定,然后利用末端快速扩增技术(RACE)对筛选到的功能基因片段进行全长扩增。结果表明构建的cDNA表达文库其初级库容量约为1.0×106 PFU,扩增文库为3.5×109 PFU;通过免疫学筛选,获得50个阳性克隆。测序和Blast分析后,鉴定出10个莫氏巴贝斯虫基因片段,RACE扩增获得了其中8个基因的全长。本试验构建的莫氏巴贝斯虫裂殖子的cDNA表达文库和筛选到的10个结构和功能基因,为研究巴贝斯虫生物学特性、筛选疫苗与诊断用抗原及药物靶位奠定了基础。

The objective of this study was to obtain functional genes of Babesia motasi, a cDNA expression library of the merozoites was constructed and immunoscreened with positive sera from sheep infected with B. motasi. The merozoites of B. motasi were purified from red blood cell with differential centrifugation. The mRNA was purified from extracted total RNA. Synthetized double-strand cDNA was added directional EcoR Ⅰ/Hind Ⅲ linkers and ligated to the EcoR Ⅰ / Hind Ⅲ arms of X screen vector. To produce a primary cDNA library of B. motasi, the phages DNA was packaged in vitro and transfected into ER1647. The positive clones were obtained by immunoscreening with the positive sera against B. motasi and amplified with rapid amplificationof cDNA ends （RACE）. The titers of the primary and amplified cDNA expression library were 1.0×10^6 PFU and 3.5 ×10^9 PFU · mL-1 , respectively. The results showed that 10 genes of B. motasi were identified and the full-length of 8 genes were amplified by RACE. The cDNA expres- sion library and genes screened provide an important material for screening and identifying candi- date antigens of vaccination and diagnosis, drug targets as well studying biological characteristics of Babesia.