长角血蜱MESK基因的克隆及其生物学特性分析  

Cloning and biological characteristics of MESK gene of Haemaphysalis longicornis

作　　者：赵波[1] 刘光远[1] 罗金[1] 张萍[1] 田占成[1] 谢俊仁[1] 田美媛[1] 王芳芳[1] 袁小松[1] 

机构地区：[1]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室甘肃省动物寄生虫病重点实验室农业部草食动物疫病重点开放实验室,甘肃兰州730046

出　　处：《中国兽医科学》2013年第8期806-811,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31201899);甘肃省自然科学基金项目(096RJZA128)

摘　　要：为进一步了解MESK基因在长角血蜱免疫应答中的作用,克隆了长角血蜱MESK基因全长序列,采用有关生物信息学软件对该基因序列的一级结构进行了分析;构建了真核表达载体pEGFP-C1-MESK,采用脂质体转染法在HEK293细胞及HeLa细胞中对其进行了表达。48h后对细胞进行裂解,用兔抗长角血蜱阳性血清与表达出的重组蛋白进行反应,以检测重组蛋白的免疫活性。结果显示,MESK基因全长为1 426bp,包括1个1 185bp的开放阅读框。该基因含有5个N-糖基化位点、5个豆蔻酰化位点、9个酪氨酸蛋白Ⅱ激酶磷酸化位点、1个酪氨酸蛋白激酶位点、2个cAMP和cGMP依赖性蛋白激酶磷酸化位点、1个蛋白磷酸激酶C磷酸化位点。SDS-PAGE分析及Western-blot分析表明,表达出的重组蛋白的分子质量约为71ku,且以与兔抗长角血蜱阳性血清反应,免疫原性良好。结果表明,MESK基因可以作为一种重要的分子标记参与蜱分类的研究,在作为候选抗蜱疫苗的研究中具有重要的开发价值。To further investigate the immune response of MESK gene in Haemaphysalis longicornis,the full-length cDNA sequence of MESK gene of Haemaphysalis longicornis was cloned and analyzed by the bioinformatic methods.Eukaryotic expression was successful conducted in HEK293 cells and HeLa cells by liposome transfection. The immunological competence of MESK protein was detected with the horseradish peroxidase-labeled donkey anti-rabbit antibody by Western-blotting. In result,the full-length cDNA of Haemaphysalis longicornis was 1 185 bp in size,containing a 1 185 bp open reading frame（ORF）. MESK was predicted to have 5 N-glycosylation sites,5 N-myristoylation sites,9 tyrosine protein Ⅱ kinase phosphorylation sites,1 tyrosine protein kinase,2 cAMP-and-cGMP dependent protein kinase phosphorylation sites,and 1 protein kinase C phosphorylation site. The expressed fusion protein was 71 ku in a molecular weight,and had immunogenicity by Western-blotting. In conclusion,the MESK gene as an important molecular marker may play an important role in tick classification as well as a candidate gene for anti-tick vaccine researches.

关 键 词：长角血蜱 MESK基因 生物信息学 克隆 真核表达 

分 类 号：S852.746[农业科学—基础兽医学]