摘要
目的 探讨恶性疟原虫富组蛋白Ⅱ (Histidine richproteinⅡ ,HRPⅡ )基因在真核细胞中的表达。方法 应用聚合酶链反应 (PCR)扩增HRPⅡ全编码区基因 ,经HindⅢ和BamHI双酶切后定向克隆入带CMV启动子的真核高效表达载体 pcDNA3,构建HRPⅡ /pcDNA3重组载体 ;用脂质体法将重组载体转染HepG2肝癌细胞 ,经G418加压筛选后 ,收集阳性克隆细胞培养上清 ,表达产物进行十二烷基硫酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)和Western免疫印迹分析。结果 成功构建HRPⅡ / pcDNA3真核表达载体 ,SDS PAGE和Western免疫印迹分析表明 ,HRPⅡ表达产物相对分子质量约 35 0 0 0 ,呈分泌性表达 ,且表达产物能被抗HRPⅡ单克隆抗体特异识别。
Objective Plasmodium falciparum synthesizes and secretes histidine rich proteins Ⅱ (HRP Ⅱ) into plasma during asexual blood stage of life cycle. HRP Ⅱ is an ideal diagnostic antigen for falciparum malaria. In this report, our purpose was to investigate gene expression of HRP Ⅱ in eukaryotic cells. Methods The entire coding region of HRP Ⅱ gene was amplified by polymerase chain reaction (PCR). PCR products were cut by Hind Ⅲ and Bam HI, and inserted into eukaryotic expression vector pcDNA3. Recombinant vectors were transfected HepG2 cells by means of liposome. Positive clones were cultured and screened under the high level of G418. The supernatants of these clones were collected and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting assay. Results HRP Ⅱ gene was successfully cloned into pcDNA3 vector. The results of SDS PAGE and Western blotting assay showed:(1) the molecular weight of the expressed products was about 35 000, (2) HRP Ⅱ was secreted into culture supernatant by the signal peptide, (3) the expressed products could specifically bind with anti HRP Ⅱ McAbs. Conclusion The findings suggested that HRP Ⅱ gene of Plasmodium falciparum had been successfully expressed in pcDNA3/HepG2 eukaryotic system.
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2000年第2期110-113,共4页
Chinese Journal of Infectious Diseases
基金
国家"九五"攻关项目! ( 96 90 6 0 4 0 6)
广东省青年自然科学基金!( 960 12 3 )
