刺激隐核虫钙调蛋白基因的分子鉴定  

Molecular identification of a calmodulin gene from Cryptocaryon irritans

作　　者：倪炜[1,2] 张经伟 余玲莹 黄晓红 NI Wei;ZHANG Jingwei;YU Lingying;HUANG Xiaohong(Fujian Key Laboratory of Developmental Biology and Neuroscience,College of Life Science,Fujian Normal University,Fuzhou 350117,China;Department of Molecular Genetics and Microbiology,University of Florida,Gainesville 32610,USA)

机构地区：[1]福建师范大学生命科学学院,福建省发育与神经生物学重点实验室,福建福州350117 [2]美国弗罗里达大学分子遗传学和微生物学系,Florida Gainesville 32610

出　　处：《水产学报》2022年第5期774-784,共11页Journal of Fisheries of China

基　　金：国家自然科学基金(31672244);“十三五”海洋经济创新发展示范项目(FZHJ11)。

摘　　要：为了解钙调蛋白在刺激隐核虫生长发育过程中的作用,实验从刺激隐核虫滋养体cDNA文库中克隆出钙调蛋白基因(cicam),优化密码子后人工合成开放阅读框(ORF),构建成重组质粒pGEX-4T-1/cicam,将其转化至大肠杆菌后,通过ZYM-5052自诱导培养基诱导重组子表达。用谷胱甘肽琼脂糖凝胶及凝血酶纯化重组蛋白rCiCaM,并用其免疫小鼠BALB/c株获得多克隆抗体。分别用逆转录PCR和免疫印迹实验检测cicam及其编码蛋白CiCaM在各期虫体中的表达。通过间接荧光抗体实验检测CiCaM在幼虫中的定位。通过覆盖印迹实验(Blot overlay assay)初步探讨了重组蛋白rCiCaM结合重组肌动蛋白解聚因子的活性。结果显示,cicam的ORF为450 bp,编码含149个氨基酸的肽链,其分子量预测值为16.9 ku;原核表达的融合蛋白GST-rCiCaM及切除GST标签的rCiCaM的表观分子量分别为43和16.9 ku,均与预测值相符;cicam在刺激隐核虫的各个发育阶段都有稳定的表达,其表达产物CiCaM的分子量与预测值相符;在幼虫细胞内CiCaM在胞质中均有分布,尤其在4个大核周围及胞口部位更为富集;CiCaM与CiADF;之间可能发生Ca;依赖性的相互作用。该研究丰富了刺激隐核虫病原分子生物学知识,为刺激隐核虫病防治方法的开发提供参考。Cryptocaryon irritans is a parasitic ciliate causing‘white spot disease’on marine teleosts that often results in huge financial losses to mariculture.So far,there is no safe and effective method to control the parasite infection.Calmodulin(CaM),as a sensor for Ca;signaling,regulates cell movement,cell division and invasion of some protozoan parasites to their hosts.To understand the role of calmodulin in the growth and development of C.irritans,the calmodulin gene(cicam)was cloned from the C.irritans trophozoite cDNA library,and the open reading frame(ORF)was synthesized after codon optimization.The recombinant plasmid pGEX-4T-1/cicam was constructed,then transformed into Escherichia coli and induced its recombinant expression with ZYM-5052self-inducing medium.The recombinant protein rCiCaM was purified by glutathione agarose gel and thrombin,and obtain polyclonal antibodies by immunizing with the mouse BALB/c strain.The expression of cicam and its encoded protein CiCaM in each stage of the worm was examined by reverse transcription PCR and immunoblotting assay,respectively.The localization of CiCaM in the larvae was examined by indirect fluorescent antibody assay.The activity of recombinant protein rCiCaM binding recombinant actin-depolymerizing factor was initially investigated by blot overlay assay.The results showed that the open reading frame(ORF)of CiCaM was 450 bp,which encoded a polypeptide of 16.9 ku,consisting of 149 amino acids;the molecular masses of GST-rCiCaM and rCiCaM were 43 ku and 16.9 ku respectively,which corresponded with the predicted ones;the CiCaM gene expressed in all developmental stages of C.irritans,and the molecular mass of the native CiCaM corresponded to the predicted value;the CiCaM distributed all over the cytosol of C.irritans theronts,especially abundant around the four macro nuclei and the peripheral area of cytostome;rCiCaM could interact with rCiADF;in a Ca;-dependent way.This study enriched the knowledge of molecular biology of the pathogen C.irritans,which would provide

关 键 词：刺激隐核虫 钙调蛋白 克隆 表达 分子鉴定 

分 类 号：Q71[生物学—分子生物学] S941.51[农业科学—水产养殖]