恶性疟原虫肝素结合蛋白质PF3D71104400的原核表达与多克隆抗体的制备

Prokaryotic Expression and Preparation of Polyclonal Antibody of Heparin Binding Protein PF3D7_1104400 of Plasmodium falciparum

为研究肝素结合蛋白质PF3D7_1104400的功能以及其作为疟疾疫苗候选抗原的可行性,通过PCR方法扩增目的基因,并将其与p ET-28a和p GEX-4T-1表达载体连接构建重组表达质粒。将构建成功的重组表达质粒转化入大肠杆菌BL21-Condon Plus(DE3)-RIPL中诱导表达,利用亲和层析的方法纯化His-tag和GSTtag重组蛋白质。用His-tag重组蛋白质免疫家兔制备多克隆抗体,以该抗体为一抗,利用Western blot检测该抗体的特异性以及PF3D7_1104400蛋白质在虫体内是否表达。结果表明:成功构建重组表达质粒PF3D7_1104400-p ET和PF3D7_1104400-p GEX,诱导表达并纯化出特异性好、纯度高的重组蛋白。制备的多克隆抗体能够特异性识别虫体天然蛋白,PF3D7_1104400蛋白质在恶性疟原虫体内全长表达。

To research the biological function of heparin binding protein PF3D7_1104400 and its feasibility as candidate vaccine antigen,target gene was amplified by PCR whose products were connected with p ET-28 a and p GEX-4T-1 for constructing recombinant expression plasmids. Transformed into BL21-Condon Plus（ DE3）-RIPL,recombinant plasmids were induced and expressed and recombinant proteins were purified by the method of affinity chromatography. Polyclonal antibody was prepared by rabbits immuned with His-tagged recombinant proteins. The specificity of antibody and expression of PF3D7_1104400 were detected by Western blot in which polyclonal antibody was used as primary antibody. The results showed that recombinant plasmids,PF3D7_1104400-p ET and PF3D7_1104400-p GEX,were constructed successfully and specific recombinant proteins were expressed and purified. According to the results of Western blot,natural protein of PF3D7_1104400 could be recognized specifically by prepared antibody and was expressed in Plasmodium falciparum in the form of total length,which will lay foundation for further research of PF3D7_1104400.