中国兽医科学2018，Vol.48Issue(2)：135-141,7.DOI:10.16656/j.issn.1673-4696.2018.0021
吕氏泰勒虫TlSP基因多态性免疫区在毕赤酵母中的高效分泌表达
Efficient secretory expression of the polymorphic immunodominant region of TlSP gene of Theileria luwenshuni in Pichia pastoris
摘要
Abstract
The present study aimed to prepare Theileria luwenshuni surface protein(TlSP) with complete antigenicity and natural structure.The total RNA was extracted from T.luwenshuni merozoites and the cDNA of TlSP gene was reversely transcribed using RT-PCR.The polymorphic immunodominant region of TlSP gene was cloned into pPIC9K vector to form the recombinant plasmid pPIC9K-TlSP.The recombinant plasmid was linearized by Sal Ⅰ and then transformed into Pichia pastoris GS115 and KM71 by electroporation,and the multi-copy transformants with hyper-resistance to Geneticin G-418 were obtained by screening.The positive strains by PCR were induced by methanol.The effect of recombinant TlSP expression in GSll5 and KM71 was evaluated,and the reactionogenicity of TlSP was analyzed.The result showed that the cDNA sequence of T1SP gene was 888 bp and its polymorphic immunodominant region was 552 bp in length;5 colonies in GSl15 and 7 colonies in KM71 resistant to 4.0 mg/mL Geneticin were obtained,and the recombinant protein TlSP was secreted in the above two strains.The effect of TlSP expression in GSl15 was better than in KM71.The results showed that the recombinant TlSP could react with the positive sera against T.luwenshuni,T.uilenbergi,T.ovis and T.annulata,but not with the positive sera against Babesia spp.and Anaplasma ovis.This study laid the foundation for further research on the immunological diagnosis and the development of subunit vaccines of TlSP protein in ovine and caprine theileriosis.关键词
吕氏泰勒虫/TlSP基因/毕赤酵母/分泌表达/反应原性Key words
Theileria luwenshuni/TlSP gene/Pichia pastoris/secretory expression/reactionogenicity分类
农业科技引用本文复制引用
郭鹏飞,刘光远,刘志杰,李有全,韩元,刘军龙,刘爱红,王锦明,李禤,关贵全,殷宏,罗建勋..吕氏泰勒虫TlSP基因多态性免疫区在毕赤酵母中的高效分泌表达[J].中国兽医科学,2018,48(2):135-141,7.基金项目
国家重点研发计划项目(2017YFD0501200) （2017YFD0501200）
国家自然科学基金资助项目(31272556) （31272556）
肉牛体系专项(CARS-37) （CARS-37）
