柔嫩艾美耳球虫Cathepsin B基因的克隆表达及活性研究

Cloning,Expression and Activity of Cathepsin B in Eimeria tenella

旨在利用同源克隆法从柔嫩艾美尔球虫(Eimeria tenella)中克隆组织蛋白酶B基因(Etcat B),为进一步研究其基因表达和基因功能奠定基础。选用未孢子化的柔嫩艾美耳球虫Houghton株,通过对已公布的柔嫩艾美尔球虫序列进行分析,利用RT-PCR的方法克隆EtcatB基因的cDNA序列,并将其重组于transE1原核表达载体中,鉴定后,重组质粒转入大肠杆菌Rosetta进行诱导表达。利用纯化所得Etcat B蛋白制备兔抗多抗,同时利用明胶酶谱法初步验证其活性。利用Real-time PCR和Western blot的方法检测Etcat B在球虫发育不同阶段和孢子化不同阶段的表达水平。成功克隆得到一个长度为1 539bp的Etcat B基因,并重组表达了球虫的组织蛋白酶B。SDS-PAGE电泳结果显示,在1mmol·L-1 IPTG 37℃诱导4h下,主要以包涵体的形式表达;4℃过夜诱导可以使之转为可溶性表达。表达产物在低pH、有还原剂存在的环境中,可以降解明胶,初步证明重组表达的酶具有一定的活性,Etcat B在配子体和裂子体时期表达较高,未孢子化和子孢子阶段表达相对较低,孢子化过程中表达逐步增加。Etcat B基因的克隆和表达研究,为进一步探究该基因在鸡球虫入侵及其他生物学功能中所起的作用提供了理论基础。

The aim of the present study was to clone the cathepsin B(Etcat B)gene involved in E.tenella using homologous method,and lay the the foundation for further gene expression and gene function study.Through analysis of E.tenellasequence published,the cDNA sequence of Etcat B was cloned by RT-PCR from unsporulated E.tenella Houghton strain.The Etcat B was recombined in trans-E1 prokaryotic expression vector.After sequence identification,the recombinant plasmid was transfected into E.coli rosetta to induce expression.The purified protein was used to immunize rabbits to produce polyclonal antibody,which was verified by ELISA,and was also analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)gelatin zymography.With the method of real-time PCR and Western blot,we detected the expression of Etcat B and Etcat L at different stage.Results showed that an 1 539 bp gene was obtained,which encoded512 amino acid residues.The Etcat BcDNA fragment was sub-cloned into the prokaryotic expression vector trans-E1.After inducing in 1mmol·L-1 IPTG at 37 ℃ for 4h,SDS-PAGE electrophoresis showed it was expressed mainly in the form of inclusion bodies;while at 4 ℃ overnightthe protein was inducted into soluble expression.With the presence of reducing agent at low pH,the recombinant protein can degradate the gelatin,which proved that the recombinant enzyme having a certain activity.Etcathespin B was higher in the phase of gametophyte and merozoite,and gradually increased during sporulation process.Cloning and expression the cathepsin B in E.tenella,provides theoretical basis for further exploration of the role of the gene in chicken coccidia invasion and other biological functions.