伊氏锥虫PFR2蛋白的原核表达及多克隆抗体的制备

Prokaryotic expression and preparation of polyclonal antibody of PFR2 protein from Trypanosoma evansi

为了探究伊氏锥虫副鞭毛杆蛋白2(paraflagellar rod protein 2,PFR2)的分子特征和免疫特征,利用PCR扩增伊氏锥虫PFR2基因,将扩增产物插入p QE-80L载体,经原核表达后,将纯化后的重组蛋白免疫小鼠,制备多克隆抗体,利用Western-blot鉴定反应原性。结果表明,PFR2基因大小为1803 bp,编码含601个氨基酸残基的蛋白质,SDS-PAGE电泳显示该蛋白大小约为66 ku,主要以包涵体的形式表达。用间接ELISA方法检测小鼠抗PFR2多克隆抗体的效价为1∶25600;Western-blot检测显示重组PFR2蛋白具有较好的反应原性。本研究为进一步研究伊氏锥虫PFR2蛋白的功能奠定了基础。

In order to explore the molecular and immune characteristics of paraflagellar rod protein 2(PFR2)from Trypanosoma evansi,the PFR2 gene was amplified by ordinary PCR,and then was inserted into the p QE-80L vector.After prokaryotic expression,the purified recombinant protein was used to immune mice,and the polyclonal antibodies were prepared,and the reactivity of the recombinant protein was identified by Western-blot.The results showed that PFR2 gene was 1803 bp in length,encoding a protein with 601 amino acid residues.SDS-PAGE showed that the recombinant PFR2 was about 66 ku in molecular weight and expressed in precipitation in the form of inclusions.The mouse anti-PFR2 polyclonal antibody titer was 1∶25600detected by indirect ELISA method.Western-blot showed that the recombinant PFR2 protein had good reactivity.This study laid a foundation for further research of the function of PFR2 protein from T.evansi.