构建重组人CatSper1特异抗原原核表达载体

Construction of prokaryotic plasmid of recombinant human Catsper1-specific antigen

目的:构建重组人Catsper1特异抗原(含胞外区、钙选择孔区和破伤风类毒素通用T细胞表位TT580-599)原核表达载体并表达重组抗原。方法:设计引物,以RT-PCR法扩增人CatSper1的整个跨膜区DNA片段,利用重叠PCR方法合成重组人CatSper1特异抗原DNA片段,并插入到pET-21b和pET-21b-Trx(硫氧还蛋白)的原核表达载体。测序鉴定后转化工程菌株E.coliBL21(DE3)进行诱导表达,并纯化表达的重组蛋白。用Tricine-SDS-PAGE和Western blotting分析重组蛋白的表达情况。结果:成功构建pET-21b-Catsper1特异抗原和pET-21b-Trx-Catsper1特异抗原原核表达载体,并在BL21(DE3)中诱导表达重组蛋白。Tricine-SDS-PAGE和Western blotting鉴定表明,已获得重组人CatSper1特异抗原包涵体和纯化的可溶重组Trx-Catsper1特异抗原。结论:成功在原核表达系统表达重组人Catsper1特异抗原。

Aim：To construct the prokaryotic expression vector of recombinant human Catsper1-specific antigen（including the extracellular region,the pore region and the promiscuous T cell epitope TT^580-599） and express the recombinant antigens. Methods： The DNA fragment of human Catsper1 coding the entire transmembrane region was amplified by RT-PCR.The DNA fragment of human Catsper1-specific antigen was amplified by overlapping PCR and cloned into prokaryotic expression vector pET-21b and pET-21b-Trx.After confirmed by sequencing analysis,the recombinant plasmids were transformed into E.coli BL21（DE3）and the recombinant proteins were expressed with IPTG induction.The recombinant fusion protein was purified.The recombinant proteins were separated by Tricine-SDS-PAGE,and identified by Western blotting.Results： The prokaryotic plasmids pET-21b-Catsper1specific antigen and pET-21b-Trx-Catsper1specific antigen were successfully constructed.The recombinant proteins were expressed after the recombinant plasmids transformed to E.coli BL21（DE3）.After analysis by Tricine-SDS-PAGE and Western blotting,the inclusion bodies of recombinant human Catsper1 specific antigen and the purified soluble recombinant Trx-Catsper1 specific antigen were obtained.Conclusion： The recombinant human CatSper1 specific antigens were successfully expressed in prokaryotic system.