环形泰勒虫TaSDP基因的原核表达及TaSDP蛋白多克隆抗体的制备

Prokaryotic expression of TaSDP gene of Theileria annulata and preparation of polyclonal antibody against TaSDP

利用PCR技术从环形泰勒虫裂殖体cDNA中扩增TaSDP(Theileria annulata schizont-derived protein)基因,用获得的基因片段构建原核表达载体pET-30a(+)-TaSDP,并在大肠杆菌BL21(DE3)pLysS中进行表达,蛋白以包涵体的形式存在;融合蛋白经纯化后免疫家兔,制备多克隆抗体,并分别用ELISA和Western-blot测定抗体的效价及特异性。结果显示,获得的TaSDP基因的长度为552bp,制备的多克隆抗体不仅可与重组蛋白His-TaSDP发生反应,而且可以识别天然的虫体蛋白,效价高于1∶215。上述研究结果为开展TaSDP在细胞调控方面的研究奠定了基础。

The cDNA of Theileria annulata schizont was used as template for amplification of T.annulataschizont-derived protein(TaSDP)gene by PCR.Following the amplification,the PCR product of TaSDP gene was inserted into the expression vector pET-30a(+)and then transformed into Escherichia coli BL21(DE3)pLysS for expression.The expressed fusion protein was mainly in the form of inclusion bodies.After purification,the protein was immunized rabbits to produce polyclonal antibody.Specificity and titer of the polyclonal antibody were determined by Western-blot and ELISA,respectively.From the result,the amplified TaSDP gene was 552 bp in length.The prepared anti-TaSDP polyclonal antibody could react with both His-TaSDP fusion protein and the natural antigen,and the antibody titer was more than1∶215.In conclusion,the TaSDP gene was amplified and the relevant polyclonal antibody from rabbit was obtained,which provide the foundation for further study on the mechanism of TaSDP in cell regulation.