恶性疟原虫FCC-1/HN株裂殖子表面抗原2基因在M.smegmatis mc^2155中的表达

Expression of the merozoite surface antigen 2 gene of Plasmodium falciparum FCC-1/HN in M.smegmatis mc^2 155

目的 研究裂殖子表面抗原 2 (merozoitesurfaceantigen 2 ,MSA2 )基因重组BCG疫苗的保护作用。方法 采用电穿孔转化法将重组质粒pBCG/MSA2导入耻垢分枝杆菌 (M .smegmatis)mc2 1 55中 ,通过卡那霉素抗性筛选并经PCR鉴定的重组M .smegmatismc2 1 55培养于middlebrook 7H9broth (M7H9)培养基 ,并添加 1 0 %M7H9enrichmentADC和 0 .0 5%Tween80 ,4 8～ 72h后于 4 5℃进行 3 0min的诱导表达 ,表达产物进行十二烷基磺酸钠 聚丙烯酰胺凝胶电泳 (SDS PAGE)及Westernblot分析。结果 SDS PAGE及Westernblot分析结果均显示在相对分子质量 (Mr)约 3 1× 1 0 3的位置上可见明显的蛋白条带 ,并与MSA2基因编码序列推断的Mr 相符。结论 恶性疟原虫裂殖子表面抗原 2可在M .smegmatis中表达 。

Objective The merozoite surface antigen 2(MSA2) is a protective antigen of Plasmodium falciparum . In order to construct the recombinant Bacillus Calmette Guerin (BCG)vaccine against Plasmodium falciparum , the recombinant mycobacteria E.coli shuttle plasmid pBCG/MSA2 carrying the MSA2 gene of Plasmodium falciparum FCC 1/HN was expressed in fast growing mycobacterium, M.smegmatis mc 2 155. Methods The recombinant plasmid pBCG/MSA2 was introduced into M.smegmatis mc 2 155 by electroporation,the recombinat M.smegmatis mc 2 155 selected by knanamycin resistance and identified by PCR, was cultivated in liquid middlebrook 7H9 medium supplemented with 10% middlebrook 7H9 enrichment ADC and 0.05% Tween 80 (M ADC TW broth) with shaking at 37℃. After 48 72?h cultivation, the cultures were induced at 45℃ for 30min, and its expressing product was analyzed by sodium dodecyl benzenne sulfonate polyacrylamide gel electrophoresis(SDS PAGE)and Western blot. Results The expressed MSA2 were observed by SDS PAGE and analysed by Western blot for 31kD molecules. Conclusion MSA2 gene can be expressed in M.smegmatis mc 2 155,which provides scientific evidences supporting BCG as multivalent vectoral vaccine.