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目的克隆、表达细粒棘球绦虫组织蛋白酶B(Eg Cat B)基因,并对其进行生物信息学预测和分析。方法提取细粒棘球绦虫总RNA反转录成c DNA,以此为模板扩增目的基因。利用无缝克隆技术和麦胚无细胞表达体系克隆表达Eg Cat B,用免疫印迹实验进行验证。采用Signal P 4.1、TMHMM sever v.2.0、Target P 1.1对Eg Cat B编码蛋白分别进行信号肽、跨膜区及亚细胞定位的预测。采用BLASTP和Gene Doc进行Eg Cat B同源序列比对及保守位点分析。采用Prot Param、SMART、Predictprotein、Swiss-model分析Eg Cat B编码蛋白的结构。采用Net OGlyc 4.0 Server和Net NGlyc 1.0 Server在线分析Eg Cat B蛋白O型和N型糖基化位点。结果成功构建了重组质粒p EU-Eg Cat B。蛋白电泳和免疫印迹实验结果显示该基因获得了可溶性表达。经生物信息学分析预测该蛋白分子量35.9 k Da、理论等电点6.37,为含信号肽的分泌蛋白,酶活性位点高度保守,并通过Gln106、Cys112、His282和Asn302形成了催化中心。Eg Cat B基因所编码蛋白氨基酸序列中不存在N-糖基化位点,但存在9个O-糖基化位点。结论成功克隆表达了细粒棘球绦虫Eg Cat B基因并对其进行了较全面的生物信息学预测分析,为该蛋白的功能研究提供了参考依据。
Objective To clone and express Eg Cat B gene of Echinococcus granulosus and predict its bioinformatics. Methods The total RNA of Echinococcus granulosus was extracted and reverse transcribed into c DNA, which was used as a template to amplify the target gene. Eg Cat B was cloned using a seamless cloning technique and wheat germ cell-free expression system and verified by Western blotting. The signal peptide, transmembrane region and subcellular localization of Eg Cat B protein were detected by using Signal P 4.1, TMHMM sever v. 2.0 and Target P 1.1. BLASTP and Gene Doc were used to compare Eg Cat B homology sequences and conservative site analysis. The structure of Eg Cat B encoding protein was analyzed using Prot Param, SMART, Predictprotein, Swiss-model. Eg Cat B protein O and N glycosylation sites were analyzed online using Net OGlyc 4.0 Server and Net NGlyc 1.0 Server. Results The recombinant plasmid p EU-Eg Cat B was successfully constructed. Protein electrophoresis and Western blotting results showed that the gene obtained soluble expression. Bioinformatics analysis predicted that the protein had a molecular weight of 35.9 kDa and a theoretical isoelectric point of 6.37, which was a secreted protein with signal peptide. The active site of the enzyme was highly conserved and formed a catalytic center through Gln106, Cys112, His282 and Asn302. There is no N-glycosylation site in the amino acid sequence of the protein encoded by the Eg Cat B gene, but 9 O-glycosylation sites exist. Conclusion The Eg Cat B gene of Echinococcus granulosus was successfully cloned and expressed, and a more complete bioinformatics prediction analysis was carried out, which provided a reference for the function of this protein.