东北七鳃鳗Lm-PHB2的生物信息学分析及多克隆抗体制备

Bioinformatics analysis and antibody preparation of Lm-PHB2 fromLampetra morii

在首次获得七鳃鳗Lm-PHB2的全长cDNA序列的基础上,对预测得到的LmPHB2氨基酸序列进行了生物信息学和进化分析;经原核表达并大量提纯获得重组蛋白rLm-PHB2,在此基础上,优化兔抗七鳃鳗抗体的免疫制备流程.结果表明:七鳃鳗LmPHB2蛋白的理论分子质量约为33.25ku,理论等电点为9.85,为亲水性蛋白;信号肽预测结果表明其ORF区中无信号肽.该蛋白有1个位点可能发生N型糖基化,不存在棕榈酰化位点;跨膜区预测表明Lm-PHB2仅在内膜区域存在跨膜结构.系统发育树分析表明Lm-PHB2的进化地位在头索动物与脊椎动物之间,与物种进化规律基本一致.制备了效价≥64万且与重组蛋白rLm-PHB2和天然Lm-PHB2蛋白均有很强的特异性结合能力的多克隆抗体.Western Blot表明,Lm-PHB2蛋白在七鳃鳗的心、肾、肝和肠中均有表达,其中,在心脏组织中表达量最高.本实验为PHB2基因的进化研究提供了理论依据,并为七鳃鳗Lm-PHB2蛋白功能研究奠定了基础.

Based on sequencing the open reading frame of Lm-PHB2 complement DNA firstly,the characterization of Lm-PHB2 was studied by the bioinformatics and phylogenetic analysis.The recombinant Lm-PHB2 protein was purified abundantly with the prokaryotic expression system before optimizing the immunization protocol within the preparation of rabbit anti-Lm-PHB2.The predicted molecular weight of Lm-PHB2 is 33.24 kD with the theoretical isoelectric point of 9.85.It is a hydrophilic protein with the grand average hydropathicity at-0.207.It has no signal peptide and no site of palmitoylation,and only 1site which may be motified by N-glycosylation appears within the protein.Lm-PHB2 is not a transmembrane protein due to the sequence of its amino acid having no transmembrane regions.It was shown from the phylogenetic analysis that L.morii was between the cephalochordate and the vertebrate,which was consistent with the law of evolution.It demonstrated that the specificity of the polyclonal antibody to the rLm-PHB2 and Lm-PHB2 was very high and the titer was higher than 640 thousand.Western Blotting showed that Lm-PHB2 was detected in heart,kidney,liver and intestine,and the amount was highest in heart.This study may lay the foundation for the further investigation of Lm-PHB2 about evolution and Lm-PHB2 concerning functions.