杜氏利什曼原虫无鞭毛体表达抗原的Westernblot和MALDI-TOF／TOF分析

Identification of the specific antigen expressed by Leishmania donovani amastigotes

目的鉴定体氏利什曼原虫无鞭毛体特异表达抗原。方法培养杜氏利什曼原虫前鞭毛体并体外转化无鞭毛体，其总蛋白经2-DE电泳后以小鼠抗杜氏利什曼原虫无鞭毛体血清进行Western blot，对前鞭毛体与无鞭毛体特砰表达抗原蛋白进行MALDI-TOF／TOE串联质谱鉴定。重组表达无鞭毛体特异表达抗原编码基因，以Westernblot法对重组蛋白进行鉴定。结果等量的杜氏利什曼原虫前鞭毛体与无鞭毛体蛋白经2-DE电泳均可呈现680～742个蛋门点，Westernblot及MALDI-TOF／TOFMS分析甘油醛3-磷酸脱氢酶与延伸因子2为杜氏利什曼原虫前鞭毛体特异表达抗原，核甘二磷酸激酶为无鞭毛体特异表达抗原。重组核苷二磷酸激酶编码基因表达产物经Westernblot证实为朴氏利什曼原虫无鞭毛体特异表达强抗原。结论杜氏利什曼原虫前鞭毛体与无鞭毛体抗原表达存存差异，核甘二磷酸激酶为杜氏利什曼原虫无鞭毛体特异表达强抗原。

Objective To identify the specific antigen expressed in Leishmania donovani amastigotes. Methods The promastigotes of L. donovani were cultured and transferred into axenie amastigotes in vitro. The total proteins of pro mastigotes and axenic amastigotes were separated using two dimensional electrophoresis （2 DE） in a broad pI range （3 10）. Proteins were identified by Western blotting with mouse serum against L. donovani amastigotes. Antigens specific ally expressed by promastigotes and axenic amastigotes were analyzed with MALDI-TOF/TOF-MS. The gene encoding a specific antigen expressed by amastigotes was cloned and expressed in E. coli, and the recombinant protein was identified with Western blotting. Results 2-DE revealed approximately 680--742 protein spots for corresponding proteins of pro- rnastigotes and axenic amastigotes. Proteins were analyzed using Western blotting and MALDI TOF/TOF MS. Glyceral dehyde 3-phosphate dehydrogenase and elongation factor 2 were identified as antigens specifically expressed by promastig- otes, and nueleoside diphosphate kinase was identified as an antigen specifically expressed by amastigotes. The recombi- nant nucleoside diphosphate kinase was identified as a strong antigen specific to L. donovaniamastigotes according to Western blotting. Cenelusion These findings demonstrate the differences in the antigen expression profiles of promas tigotes and amastigotes. Nucleoside diphosphate kinase is an antigen specifically expressed by amastigotes.