恶性疟原虫裂殖子新型蛋白质(PF3D7_0811600)的表达纯化及其多克隆抗体的制备  

Expression and Purification of the New Merozoite Surface Protein PF3D7_0811600 and Preparation of Polyclonal Antibody

作　　者：周健华[1] 魏晓燕[1] 常志广[1] 姜宁[1] 陆慧君[1] 陈启军[1] 

机构地区：[1]吉林大学人兽共患病研究所,人兽共患病教育部重点实验室,长春130062

出　　处：《吉林农业大学学报》2016年第2期218-222,共5页Journal of Jilin Agricultural University

基　　金：国家自然科学基金项目(81420108023)

摘　　要：利用大肠杆菌原核表达并纯化恶性疟原虫3D7株裂殖子新型蛋白质PF3D7_0811600,免疫家兔制备特异性多克隆抗体,通过对虫体天然蛋白的识别,分析PF3D7_0811600在虫体内的表达。采用PCR技术从恶性疟原虫3D7株总DNA中获得PF3D7_0811600基因的目的片段,连接到p MD18-T克隆载体,得到的克隆质粒经EcoRⅠ和XhoⅠ双酶切后,构建重组原核表达质粒p ET-PF3D7_0811600和p GEX-PF3D7_0811600,通过优化表达条件,分别在大肠杆菌中得到高效表达,并用亲和层析柱纯化目的蛋白质。用纯化的His标签重组蛋白质免疫家兔,用Western Blot方法检测制备的特异性多克隆抗体。结果表明:成功纯化并获得重组蛋白质,用间接ELISA方法检测多抗效价达到1∶16 000,用Western Blot检测结果表明制备的多克隆抗体具有良好的特异性,能够识别虫体天然蛋白。恶性疟原虫3D7株裂殖子新型蛋白质PF3D7_0811600在虫体晚期表达,说明该蛋白在虫体发育的晚期发挥重要作用,猜测其可能与裂殖子入侵宿主红细胞有关。A new merozoite surface protein PF3D7_0811600 was expressed by E.coli,then purified by either HIS system or GST system. The recombinant protein was used to immune rabbits to develop the special polyclonal antibody which identified the natural protein of parasite and other analysis.The fragment of PF3D7_ 0811600 gene was amplified from total DNA by PCR,then cloned to p MD18-T,and the positive clone plasmid was obtained. After restriction enzyme digestion by EcoRⅠ and XhoⅠ,recombinant plasmid p ET-PF3D7_0811600 and p GEX-PF3D7_0811600 were constructed in E.coli respectively. Polyclonal antibody was raised in Rabbits by immunizing with purified His-tagged recombinant protein and analyzed by Western Blot. The recombinant protein was successfully purified,and the titer of polyclonal antibody was as high as 1 ∶ 16 000 detected by indirect ELISA assay. The results of Western Blot showed that polyclonal antibody against PF3D7_0811600 is highly specific,and it is able to identify natural protein of parasite. The new merozoite surface protein PF3D7_0811600 is expressed in the late stage of parasite,and PF3D7_ 0811600 plays an important role in the late stage of parasite and may participate in the merozoite invading red blood cells.

关 键 词：恶性疟原虫 裂殖子 原核表达 多克隆抗体 

分 类 号：S855.99[农业科学—临床兽医学]