曼氏迭宫绦虫钙/钙调素依赖蛋白激酶I(SmCaMK I)基因的生物信息学分析与表达鉴定

Sequence bioinformatics analysis,expression and identification of Calcium/Calmodulin-dependent protein kinase I(CaMK I)from Spirometra mansoni

目的开展SmCaMK I(曼氏迭宫绦虫钙/钙调素依赖蛋白激酶I)的潜在生物学特征分析和功能研究。方法利用相关网站和软件对SmCaMK I的同源性核苷酸序列比对分析、保守位点预测、构建分子进化树、编码氨基酸的淋巴细胞表位进行分析预测。此外,SmCaMK I进行扩增,并克隆到表达载体pET-28α(+)中进行蛋白的表达纯化,制备大鼠免疫血清进行虫体免疫组织定位分析。结果 SmCaMK I是一个全长基因,由1 068bp组成,编码355个氨基酸。SmCaMK I与细粒棘球绦虫、多房棘球绦虫的CaMK I保守功能域的氨基酸序列一致性分别为89%和88%,与人的CaMK I氨基酸序列一致性仅仅只有44%。分子进化树分析中SmCaMK I与绦虫属的亲缘关系最近,与其他物种亲缘性较远。与人类CaMK I相比,淋巴细胞表位具有统计学差异。SmCaMK I能够定位在成虫的睾丸和虫卵,在裂头蚴中大量表达和特异定位于体表。结论SmCaMK I是参与虫体生长发育繁殖的重要蛋白分子,还可能是一个潜在的疫苗靶标蛋白。

The aim for this study is predicting the biological characteristics and potential functions of Calcium/calmodulin dependent protein kinase I（CaMK I）fromSpirometra mansoni（SmCaMK I）,and cloning it into prokaryotic expression vector,as well as performing immunohistochemical localization,which paved the way for the further study.The nucleotide sequence homology analysis,the conserved sites prediction the physical and chemical parameters and the lymphocyte epitope of SmCaMK I were performed by NCBI website and online software.The evolution tree was built by MEGA5.0software.In addition,the CaMK I genes was amplified and cloned into the expression vector pET-28α（＋）.The protein was expressed and purified,as well as used to prepared for anti-rSmCaMK I rat serum,which was used to perform immunohistochemical localization in parasites.SmCaMK I was a full length gene,composed of 1 068 bp and encoded 355 amino acids.Whats more,the deduced amino acid sequence shared above 88%identities with Echinococcus granulosus and Echinococcus multilocularis,but 44%identity with Homo sapiens.The analysis of system evolution tree showed that the genetic relationship between SmCaMK I was the closest and CaMK I from Taenia and far from other species.The lymphocyte epitope prediction showed obviously different between SmCaMK I and CaMK I of Homo sapiens.The western Blot results indicate that SmCaMK I has great immunogenicity.Immunohistochemical localization detection showed that SmCaMK I was expressed in the testis and eggs of adult worms,and also extensive distributed in tissue and tegument of sparganum.The study indicated that SmCaMK I is a critical protein involving in parasites growth and reproduction,and may be a potential vaccine target protein.