莫氏巴贝斯虫滑动体组件蛋白基因gap45、gap50、myo A和mlc的克隆表达与多克隆抗体制备

Cloning and Expressing Glideosome Associated Protein Genes of Babesia motasi and Preparation of Their Polyclonal Antibodies

【目的】克隆和表达莫氏巴贝斯虫滑动体组件蛋白GAP45(gliding associated protein 45,GAP45)、GAP50(gliding associated protein 50,GAP50)、肌球蛋白A(Myosin A,Myo A)和肌球蛋白轻链(myosin lightchain,MLC)基因及制备兔源性多克隆抗体。【方法】利用末端快速扩增(rapid-amplification of cDNA ends,RACE)技术从莫氏巴贝斯虫裂殖子cDNA中扩增gap45、gap50、myo A及mlc基因,将获得的全长或基因的部分序列构建pGEX-4T-1原核表达载体,在大肠杆菌BL21(DE3)pLys中用IPTG诱导表达;融合蛋白经纯化后免疫家兔,制备多克隆抗体,并用ELISA测定抗体的效价及反应性。【结果】获得了gap45、gap50、myo A和mlc基因的全长分别为664、1391、773和2 538 bp,开放阅读框长度分别为567、1194、606和2 514 bp,制备的多克隆抗体具有较好的特异性,效价高于1:25 600。【结论】克隆了滑动体组件蛋白gap45、gap50、myo A和mlc基因的全长,并制备了多克隆抗体,为筛选巴贝斯虫疫苗和诊断用抗原和药物靶位、研究其生物学入侵机制奠定了基础。

【Objective】To prepare polyclonal antibodies of component genes from glideosome,gap45,gap50,myo A,and mlc were cloned and expressed in prokaryotic system.【Method】 Rapid amplification of cDNA end（RACE） was used to amplify the full lengths of gap45,gap50,myo A,and mlc.The partial or complete open reading frames（ORF） of those genes were further inserted into prokaryotic expression vector and then transformed into BL21（DE3） pLys to express by inducing with IPTG.The purified fusion proteins,with MagneGST· Protein Purification System,were injected into rabbits to produce polyclonal antibodies.Specificities and titers of the polyclonal antibodies were determined by ELISA assay.【Result】The full-length cDNAs of gap45,gap50,myo A,and mlc,were 664,1 391,2 538,773 bp and open reading frames（ORF） were 567,1 194,2 514,and 606 bp in size.The polyclonal antibodies prepared had good specificities and the titers were more than 1：25 600.【Conclusion】In the present study,the full length of four component genes of B.motasi glideosome were amplified and their polyclonal antibodies were obtained,which will provide important biological materials for screening and identifying candidate antigens of vaccination and diagnosis,drug targets as well studying invasion mechanism of Babesia in the future.