家蚕微孢子虫海藻糖合成酶基因Nbtps1的克隆与原核表达

发布时间：2018-05-14 17:17

本文选题：家蚕微孢子虫 + 海藻糖合成酶　；参考：《蚕业科学》2017年05期

【摘要】：海藻糖在保护细胞质膜和生物大分子空间结构,维持胞内渗透压增大的过程中发挥重要作用,海藻糖合成酶是海藻糖合成途径中的一个关键酶。通过检索微孢子虫基因组数据库发现,家蚕微孢子虫(Nosema bombycis)基因组中含有4个海藻糖合成酶基因,其中Nbtps1基因的开放阅读框长1 386 bp,为单外显子结构,编码461个氨基酸残基,预测蛋白质分子质量53.7 k D,等电点5.19。Nb TPS1蛋白序列含有1个糖基转移酶结构域和4个潜在的N-糖基化位点,亚细胞定位预测该蛋白质位于细胞质中。多重序列比对分析结果表明,Nb TPS1与东方蜜蜂微孢子虫(Nosema ceranae)、兔脑炎微孢子虫(Encephalitozoon cuniculi)海藻糖合成酶的氨基酸序列相似度分别为59.0%、54.7%,系统发育进化树显示三者的亲缘关系与之相符。构建重组表达载体Nbtps1/p ET-28a(+)并转化大肠埃希菌Rosetta(DE3)菌株,经IPTG诱导表达及亲和纯化后获得最终质量浓度为1.18 mg/m L的重组融合蛋白His-Nb TPS1。用纯化后的融合蛋白制备兔多克隆抗体Anti-Nb TPS1,利用间接ELISA法测定多克隆抗体的效价达1∶25 600,并通过Western blotting检测验证了该多克隆抗体的特异性,为研究Nb TPS1的生物学功能以及家蚕微孢子虫的间接免疫检测奠定了实验基础。
[Abstract]:Trehalose plays an important role in protecting cytoplasmic membrane and biological macromolecular spatial structure and maintaining the increase of intracellular osmotic pressure. Trehalose synthase is a key enzyme in trehalose biosynthesis pathway. By searching the genome database of Microsporidium, we found that there are four trehalose synthase genes in the genome of Bombyx mori Nosema bombycis. the open reading frame of Nbtps1 gene is 1 386 BP, which is a single exon structure and encodes 461 amino acid residues. The predicted molecular weight of the protein was 53.7 KD, the isoelectric point 5.19.Nb TPS1 protein sequence contained one glycosyltransferase domain and four potential N-glycosylation sites, and the subcellular localization predicted that the protein was located in the cytoplasm. The results of multiple sequence alignment analysis showed that the amino acid sequence similarity of the trehalose synthase between Nb TPS1 and Nosema ceranaeae, Encephalitozoon cuniculil, Encephalitozoon cuniculide was 59.00.The phylogenetic tree showed that the phylogenetic tree was consistent with the phylogenetic tree. The recombinant expression vector Nbtps1/p ET-28a () was constructed and transformed into Escherichia coli strain Rosetta-DE3. The recombinant fusion protein His-Nb TPS1 with final mass concentration of 1.18 mg/m L was obtained by IPTG induction and affinity purification. Rabbit polyclonal antibody Anti-Nb TPS1 was prepared from purified fusion protein. The titer of polyclonal antibody was 1:25 600 by indirect ELISA assay. The specificity of the polyclonal antibody was verified by Western blotting detection. The results provided an experimental basis for the study of the biological function of NB TPS1 and the indirect immunoassay of Bombyx mori microsporidium.
【作者单位】：云南省农业科学院蚕桑蜜蜂研究所;江苏科技大学生物技术学院;中国农业科学院蚕业研究所;
【基金】：现代农业产业技术体系建设专项(No.CARS-22) 国家自然科学基金项目(No.31560675)
【分类号】：S884

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