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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 investigate the function of heparin-binding protein PF3D7_1104400 and its feasibility as a candidate antigen for malaria vaccine, the target gene was amplified by PCR and ligated with pET-28a and pGEX-4T-1 expression vectors to construct recombinant expression plasmids. The constructed recombinant expression plasmid was transformed into E. coli BL21-Condon Plus (DE3) -RIPL to induce expression, and His-tag and GSTtag recombinant proteins were purified by affinity chromatography. The recombinant protein of His-tag was used to immunize rabbits to prepare polyclonal antibody. The antibody was used as primary antibody. The specificity of the antibody and the expression of PF3D7_1104400 protein in the parasite were detected by Western blot. The results showed that the recombinant plasmid PF3D7_1104400-p ET and PF3D7_1104400-p GEX were constructed successfully and the recombinant protein with high specificity and purity was induced and purified. The prepared polyclonal antibody can specifically recognize the native protein of the parasite, and the PF3D7_1104400 protein is expressed in the whole length of the Plasmodium falciparum.