家蚕微孢子虫海藻糖酶(Trehalase)基因的生物信息学分析及原核表达和多克隆抗体制备

Bioinformatic Analysis,Prokaryotic Expression and Polyclonal Antibody Preparation of Nosema bombycis Trehalase Gene

海藻糖是家蚕微孢子虫(Nosema bombycis,Nb)成熟孢子的主要糖类物质之一,海藻糖酶作为海藻糖代谢的主要催化酶,在微孢子虫的发芽及侵染过程中发挥重要作用。通过生物信息学分析发现,家蚕微孢子虫的海藻糖酶具有4个序列相似度较高的编码基因拷贝(Nb Tre1、Nb Tre2、Nb Tre3和Nb Tre4),除了Nb Tre4编码蛋白质的N端有18个氨基酸组成的信号肽,其他拷贝的编码蛋白质均没有信号肽结构域,但都具有少数的N-糖基化位点,而没有O-糖基化位点,此外,丝氨酸磷酸化位点的比率也较高,二级结构较为简单,仅有螺旋区和低复杂度区。基于生物信息学分析结果,设计特异性引物克隆了Nb Tre1基因,该基因片段长933 bp,编码310个氨基酸,预测蛋白质分子质量36.7 k D,蛋白质的氨基酸序列有2个潜在的N-糖基化位点和14个磷酸化位点。通过构建重组表达载体并转化Escherichia coli Rosetta(DE3)感受态细胞,获得Nb Tre1蛋白的表达菌株,利用IPTG诱导获得大量以包涵体形式表达的融合Nb Tre1蛋白,其分子质量与预期值一致。融合Nb Tre1蛋白经亲和层析纯化后免疫新西兰白兔制备多克隆抗体,利用间接ELISA法测定多克隆抗体的效价达1∶25 600。通过Western blot检测验证了该多克隆抗体的特异性,制备的多克隆抗体能够应用于家蚕微孢子虫海藻糖酶的检测等。

Trehalose is the major carbohydrates in mature spores of silkworm microsporidium( Nosema bomby-cis,Nb). As the main enzyme to catalyze trehalose metabolism,trehalase plays an important role in spore germination and infection process of Nosema bombycis. Bioinformatic analysis showed that Nosema bombycis trehalase is coded by four gene copies( Nb Tre1, Nb Tre2, Nb Tre3 and Nb Tre4),which have high sequence identity with each other. Except for protein coded by Nb Tre4 that has an 18 amino acid signal peptide at its N-terminus,proteins coded by other three gene copies have no signal peptide. All four proteins have a few N-glycosylation sites but no O-glycosylation site,and a high number of serine phosphorylation sites. These proteins have relatively simple secondary structure,which is only composed of helical regions and low-complexity region. Based on bioinformatic analysis result,specific primers were designed and used to clone Nb Tre1 gene. The obtained gene fragment is 933 bp in length and encodes 310 amino acids.The encoded protein is predicted to have a molecular mass of36. 7 k D,2 N-glycosylation sites,and 14 phosphorylation sites.Through construction of recombinant expression vector and transformation of it into E. coli Rosetta( DE3) competent cells,a bacterial strain capable of expressing Nb Tre1 protein was obtained. A high quantity of Nb Tre1 fusion protein was obtained in inclusion body form through induction with IPTG. Nb Tre1 fusion protein has the expected molecular weight. After purification by affinity chromatography,the fusion protein was used to immunize New Zealand white rabbits for preparing polyclonal antibody. Indirect ELISA assay indicated that the prepared polyclonal antibody had a titer of 1 ∶ 25 600. Western blot analysis verified specificity of the prepared polyclonal antibody,indicating that this polyclonal antibody could be used to detect Nb Tre1 protein.