恶性疟原虫复合抗原重组真核表达质粒的构建与表达  

Construction and Expression of an Eukaryotic Recombinant Plasmid from a Hybrid Gene of Antigen for Plasmodium falciparum

作　　者：姚朗[1] 李英杰[1] 李明[1] 

机构地区：[1]第一军医大学热带军队卫生学系疟疾免疫室,广州510515

出　　处：《中国寄生虫学与寄生虫病杂志》2002年第2期76-78,共3页Chinese Journal of Parasitology and Parasitic Diseases

摘　　要：目的　构建编码恶性疟原虫不同发育期抗原表位的复合抗原基因HGFSP的重组真核表达质粒。　方法　将HGFSP亚克隆于真核表达载体pcDNA3构建重组真核表达质粒 pc HGFSP ;琼脂糖凝胶电泳和限制性内切酶分析鉴定 ;脂质体介导转染HepG2细胞。取经G418筛选的阳性细胞克隆进行免疫学鉴定。 　结果　酶切及电泳鉴定证实pc HGFSP含有HGFSP。间接免疫荧光试验 (IFAT)、SDS PAGE及Westernblotting结果显示 ,pc HGFSP转染细胞含有可与HGFSP原核表达蛋白免疫血清产生特异性免疫反应的蛋白 (包含恶性疟原虫MSA1、MSA2、RESA和CSP中的抗原表位 ) ,其分子量与按HGFSP长度推算的蛋白分子量接近。　结论　用HGFSP成功构建恶性疟原虫复合抗原真核表达质粒 pc HGFSP ,其表达产物具免疫反应性。Objective To construct an eukaryotic recombinant plasmid with HGFSP, a hybrid gene encoding the antigen epitopes of MSA1, MSA2, RESA and CSP in different developing stages of Plasmodium falciparum(P.f. ). Methods HGFSP was sub-cloned into an eukaryotic expression plasmid pcDNA3 from a prokaryotic recombinant plasmid pSK-HGFSP to construct the eukaryotic recombinant expression plasmid pc-HGFSP. The identified recombinant was then transfected into HepG2 cells with liposome-mediated method. The G418-selected positive cell clones were tested to identify the immunogenicity of HGFSP-expressing antigen. Results It was evidenced that HGFSP was correctly inserted into pcDNA3 by restriction enzymes map analysis. HGFSP-expressing antigen-specific fluorescent response was observed in pc-HGFSP-transfected HepG2 cells. The results of SDS-PAGE and Western-blotting showed that there was a 23 kD protein band, which can be specifically recognized by anti-sera of HGFSP-expressing protein in pc-HGFSP-transfected HepG2 cell lysis. Conclusion Pc-HGFSP, an eukaryotic recombinant plasmid encoding hybrid antigen epitopes of P.f ., was constructed successfully and the antigenicity of pc-HGFSP-expressing protein was confirmed.

关 键 词：复合抗原 重组真核表达 质粒 构建 恶性疟原虫 DNA疫苗 基因表达 

分 类 号：R382.31[医药卫生—医学寄生虫学]