恶性疟原虫乳酸脱氢酶基因的克隆表达及表达产物免疫原性的鉴定  

Cloning and Expression of the Lactate Dehydrogenase Gene of Plasmodium falciparum and the Immunogenicity Characterization of its Expressed Product

作　　者：邱少富[1] 朱虹[1] 檀华[1] 宋立华[1] 何君[1] 端青[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2007年第6期897-900,共4页Letters in Biotechnology

摘　　要：目的:克隆表达恶性疟原虫(Plasmodium falciparum,p.f)海南株(FCC1/HN)乳酸脱氢酶(LDH),并对其免疫原性进行鉴定,为制备抗LDH抗体用于胶体金法快速检测疟原虫奠定基础。方法:应用PCR技术对恶性疟原虫乳酸脱氢酶(LDHpf)基因进行特异性扩增,将扩增产物克隆入表达载体pET-32a(+),重组表达载体经鉴定后诱导表达;重组LDHpf(rLDHpf)经纯化后,免疫家兔制备兔抗rLDHpf免疫血清,间接免疫荧光和Western印迹鉴定表达产物的免疫原性。结果:构建了pET-32a/LDHpf重组表达载体,测序后同源性分析显示p.f不同株间LDH氨基酸序列同源性大于98%,不同种间同源性也在90%以上;间接免疫荧光和Western印迹分析显示rLDHpf具有较好的免疫原性。结论:LDHpf基因高度保守;rLDHpf得到高效表达并具有良好的免疫原性。Objective： To clone and express the lactate dehydrogenase（LDH） gene of Plasmodium falciparum（p.f） FCC1/ HN isolate and to identify the immunogenicity of the recombinant protein. Methods： The LDH gene of p.f was specifically amplified by PCR and cloned into prokaryotic expression vector pET-32a（＋）, the recombinant plasmid was transferred into competent E.coli BL21（DE3）, positive clones were screened and identified by direct colony PCR, restriction enzyme digestion and sequencing. Then the recombinant vector was induced and expressed in E.coli BL21（DE3）. Rabbits were immunized with the purified protein and the anti-LDH immunosera were prepared. Indirect immunofluorescence assay and Western-blot was carried out to assess the immunogenicity of the recombinant protein. Results： The recombinant expression vector pET-32a/LDHpf was successfully constructed. DNA sequence analysis indicated the homology of LDH genes of different p.f isolates reached more than 98%, and of different species of plasmodium was also up to 90%. Indirect immunofluorescence assay and Western-blot analysis showed the recombinant protein exhibited a better immunogenicity. Conclusion： Sequence analysis showed the LDH gene of p.f was quite conserved and the recombinant protein was highly expressed in the E.coli BL21 and indirect immunofluorescence assay and western-blot analysis indicated that the recombinant protein had a good immunogenicity.

关 键 词：恶性疟原虫 乳酸脱氢酶 免疫原性 同源性 

分 类 号：Q78[生物学—分子生物学] Q959.115.4