论文部分内容阅读
目的 在 Ｈｅ Ｌａ 细胞中表达恶性疟原虫红细胞结合蛋白 Ｅ Ｂ Ａ－ １７５ 和富组氨酸蛋白 Ｈ Ｒ Ｐ－ Ｉ Ｉ， 进一步研究其生物学功能和免疫学特性。方法 将 Ｅ Ｂ Ａ－ １７５ 和 Ｈ Ｒ Ｐ－ Ｉ Ｉ 部分基因串接插入 Ｐ Ｃ Ｄ Ｎ Ａ３ 真核表达载体， 获得重组表达质粒 Ｐ Ｃ Ｄ Ｎ Ａ３ Ｅ Ｂ Ａ１７５ － Ｈ Ｒ Ｐ Ｉ Ｉ。采用磷酸钙－ Ｄ Ｎ Ａ 共沉淀法将重组质粒转染 Ｈｅ Ｌａ 细胞进行体外表达， 对表达产物进行 Ｓ Ｄ Ｓ－ Ｐ Ａ Ｇ Ｅ、 Ｄｏｔ－ Ｅ Ｌ Ｉ Ｓ Ａ 和 Ｗｅｓｔｅｒｎ － ｂｌｏｔ 鉴定。结果 表达产物占表达蛋白总量的１６４ ％ ， 相对分子量与预设计的４８ｋ Ｄａ 基本相符。表达产物与鼠抗恶性疟原虫血清及构建的重组质粒免疫鼠血清产生特异免疫反应。结论 表达载体 Ｐ Ｃ Ｄ Ｎ Ａ３ 在 Ｈｅ Ｌａ 细胞内可表达出具有一定免疫学活性的恶性疟原虫重组抗原蛋白。
Objective To express Plasmodium falciparum erythrocyte binding protein E B-175 and histidine-rich protein H R P-I I in He La cells for further study of its biological functions and immunological properties. Methods E B A-175 and H R P-I I genes were inserted into P D N A3 eukaryotic expression vector in series to obtain the recombinant expression plasmid P C D N A3 E B A175 - H R P I I. The recombinant plasmids were transfected into HeLa cells by calcium phosphate - D N A coprecipitation method. The expressed products were identified by SDS-PAGE, Dot-ELISA and Western-blot. Results The expressed product accounted for 16.4% of the total protein, the relative molecular weight and pre-designed 48k Da basically consistent. The expression product and mouse anti-Plasmodium falciparum serum and the constructed recombinant plasmid immunized rat serum to produce a specific immune response. Conclusion The expression vector P C D N A3 can express recombinant immunophenotype antigen of Plasmodium falciparum in HeLa cells.