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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- EL ISA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。
Objective: To express Plasmodium falciparum merozoite surface antigen MSA1-R2 efficiently in Escherichia coli and further study its biological function. Methods: MSA1-R2 gene was recombined in p WR4 50 I galactosidase fusion protein expression vector and transformed into E. coli. The recombinant clones were identified by restriction enzyme digestion. The expression of MSA1-R2 fusion protein was induced by IPTG. The expressed product was identified by SDS-PAGE, β-galactosidase activity, dot-ELISA and Western-blot. Results: The expressed product accounted for 35% -400% of the total bacterial protein, the relative molecular weight of 70 kDa, and galactosidase - MSA1R2 fusion protein theoretical molecular weight. After IPTG induction for 4 h, the β-galactosidase activity increased by 10-fold to 14-fold. The expressed product was confirmed to have the Plasmodium falciparum antigen epitope by dot-ELISA and Western-blot. Conclusion: p WR4 50 - E.coli expression system can express immunocompetent Plasmodium protein efficiently.