家蚕微孢子虫蛋白水解酶的活性检测及质谱鉴定和序列分析

Activity Detection,Mass Spectrometric Identification and Sequen-ces Analysis of Proteolytic Enzymes from Nosema bombycis

微孢子虫的蛋白水解酶类不仅参与代谢活动,而且可能作为一种潜在的侵染宿主的毒力因子。为了研究家蚕微孢子虫蛋白水解酶的种类及蛋白质结构特点,将家蚕微孢子虫总蛋白经非变性凝胶电泳后与底物酪蛋白孵育,检测到可水解酪蛋白的蛋白酶类的活性。采用质谱法分析该类蛋白酶主要是锌离子依赖型的细胞质型亮氨酰氨肽酶和丝氨酸蛋白酶。细胞质型亮氨酰氨肽酶序列是由1 515个碱基编码的505个氨基酸组成,无信号肽,有2个潜在的锌离子结合部位,属于金属蛋白酶M17家族;丝氨酸蛋白酶具有肽酶S8结构域和跨膜域。通过序列比对发现在兔脑炎微孢子虫、蝗虫微孢子虫、蜜蜂微孢子虫、肠道微孢子虫和比氏肠道微孢子虫中均有这2种酶,且相似度较高,其进化较为保守。

Proteolytic enzymes of Nosema bombycis not only participate in metabolic process but also are potential virulence factors during infection to host.To investigate types and protein structural characteristics of N.bombycis proteolytic enzymes,the total proteins extracted from N.bombycis spores were subjected to native gel electrophoresis and then incubated with casein.The proteolytic activity was evident because clear bands corresponding to casein digestion were observed.MALDI-TOF-MS analysis revealed that the proteolytic enzymes mainly included zinc ion dependent cytosolic leucyl aminopeptidase and serine protease.The cytosolic leucyl aminopeptidase has 505 amino acids which are coded by 1 515 bases,has no signal peptide,but has two potential zinc ion binding sites,belonging to metalloprotease M17 family.The serine protease has a peptidase S8 domain and a transmembrane domain.These two enzymes are also found in genomes of Encephalitozoon cuniculi,Antonospora locustae,Nosema ceranae,Encephalitozoon intestinalis and Enterocytozoon bieneusi by sequence alignment,all of which evolved in a highly conserved mode as shown by high sequence identities between various microsporidian species.