中国寄生虫学与寄生虫病杂志 ›› 1997, Vol. 15 ›› Issue (4): 233-237.

• 实验报道 • 上一篇    下一篇

恶性疟原虫裂殖子表面抗原MSA1第二区基因在大肠杆菌中的表达

李明1; 谢毅2; 李英杰1; 任大明2; 毕惠祥1   

  1. 1 第一军医大学热带医学研究所; 2 复旦大学遗传工程国家重点实验室
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:1997-08-30 发布日期:1997-08-30

EXPRESSION OF THE SECOND REGION OF MEROZOITE SURFACE ANTIGEN-1 OF PLASMODIUM FALCIPARUM IN E.COLI

Li Ming1; Xie Yi2; Li Yingjie1; Ren Daming2; Bi Huixiang1   

  1. 1 Institute of Tropical Medicine; The First Military Medical University; Guangzhou 510515 2 State Key Laboratory of Genetic Engineering; Fudan University; Shanghai200433
  • Received:1900-01-01 Revised:1900-01-01 Online:1997-08-30 Published:1997-08-30

摘要: 目的 :在大肠杆菌中高效表达恶性疟原虫裂殖子表面抗原 MSA1- R2 ,进一步研究其生物学功能。方法 :将 MSA1- R2基因重组于 p WR4 50 I半乳糖苷酶融合蛋白表达载体中 ,转化大肠杆菌 ,酶切鉴定重组克隆。用 IPTG诱导 MSA1- R2融合蛋白的表达 ,对表达产物进行 SDS- PAGE、β-半乳糖苷酶活性、dot- ELISA和Western- blot鉴定。结果 :表达产物占菌体总蛋白的 35%— 4 0 % ,相对分子量为 70 k Da,与半乳糖苷酶 - MSA1R2融合蛋白的理论分子量相符。IPTG诱导 4 h后 ,β-半乳糖苷酶活性可增高 10倍— 14倍 ,用 dot- ELISA和 Western- blot均证实表达产物具有恶性疟原虫抗原表位。结论 :p WR4 50 -大肠杆菌表达系统可以高效表达具有免疫学活性的疟原虫蛋白。

关键词: 恶性疟原虫, 裂殖子表面抗原, 表达, 融合蛋白

Abstract: AIM:To observe high efficientexpression of the second region of merozoite surface
antigen- 1 (MSA1 - R2 ) in E.Coli for further biological studies. METHODS:Gene encoding MSA1 -
R2 of a Chinese isolate of P.falciparum was recovered from a recombinant sequencing vector M13- MSA1 R2 and cloned into p WR- 4 5 0 I,a vector for expression of fusion protein with β-
galactosidase.The recombinant vectors were transferred into E.Coli and identified by PCR and
restriction enzyme digestion. The transformed bacteria bearing pWR-MSA1R2 plasmids were induced by IPTG for production of fusion proteins. The expressed products were analyzed by SDS-PAGE, β- galactosidase activity, dot-ELISA and Western blot. RESULTS: pWR-MSA1R2 transformed bacteria produced the desired fusion proteins with a relative molecular weight of 70 kDa, representing 35% - 40% of the total bacterial proteins. The expressed protein exhibited a strong reaction with an timalarial antibodies as detected by dot-ELISA with immune sera obtained from rabbits immunized with P.falciparum. When the products were blotted with the same antisera, specific bands of 70 kDa representing MSA1R2-galactosidase fusion proteins were identified, while the products from the same bacteria without IPTG induction failed to react with the antisera. CONCLUSION: pWR450-E. Coli expression system may be used for high efficient expression of some malarial glycoprotein antigens, with satisfactory immunological activities.

Key words: Plasmodium falciparum, merozoite surface antigen- 1, expression, fusion protein