2009•Zhongguo nongye KexueRequires access
Construction of cDNA expression library of Theileria luwenshuni and immunoscreening of antigenic genes.
Youquan Li, Luo Jian-xun, Jinliang Gao, Guan GuiQuan, Ma MiLing, Liu Zhijie, Junlong Liu, Aihong Liu, Qiaoyun Ren, Dang Zhisheng, Lu Bingyi, Guangyuan Liu, Qi Bai, Hong Yin
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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.