边缘革蜱铁蛋白1的原核表达与理化特性分析

Prokaryotic expression and physicochemical characteristics analysis of ferritin 1 in Dermacentor marginatus

为探明蜱源铁蛋白1(FER1)在边缘革蜱铁代谢中的功能,本研究经PCR扩增了边缘革蜱源FER1基因编码区(CDs),测序结果显示FER1基因CDs区为519 bp;BLAST分析其与硬蜱科常见蜱FER1的氨基酸序列同源性,利用Mega 6.0软件分析了FER1基因与其他常见蜱该基因之间的系统进化关系;利用Swiss-model、Vaxijen2.0及Allertop等程序预测分析了FER1的空间结构、抗原性等特性。结果显示,FER1基因与革蜱属其他种之间进化关系较近,其编码的氨基酸序列与硬蜱科常见蜱FER1氨基酸序列之间差异很小,边缘革蜱FER1基因的CDs区编码172个氨基酸,理论等电点(pI)为5.04,该蛋白以中空的24聚体形式存在,单个亚基的抗原位点主要集中在aa8~aa12、aa80~aa93处,位于24聚体模型表面,具有抗原性。氨基酸线性片段预测FER1未显示过敏性。将FER1基因克隆至原核表达载体,构建重组质粒pET-28a-FER1,表达重组FER1蛋白(rFER1),western blot检测其免疫原性,结果显示该蛋白具有良好的反应原性,并经His-Bind镍柱亲和层析获得纯化蛋白,其大小为25 ku。本研究分析了FER1蛋白的氨基酸序列同源及FER1基因的系统进化关系,预测了该蛋白的空间结构、抗原性等特性,通过重组蛋白表达与血清学试验,推断FER1可能存在于蜱唾液腺中,说明该蛋白具有作为抗蜱疫苗候选抗原的潜力。

To explore the function of tick-derived ferritin 1(FER1)in iron metabolism in Dermacentor marginatus,PCR approach was used to amplify coding region of FER1.Sequencing results indicated the coding region of D.marginatus FER1 was519 bp.The amino acid sequence homology between FER1 gene and other common ticks was analyzed by Mega 6.0 software.The spatial structure and antigenicity of FER1 were predicted and analyzed by Swiss-model,Vaxijen2.0 and Allertop programs.The results showed that there was a close evolutionary relationship between the FER1 gene and other species of the Ixodes genus,and there was little difference between the amino acid sequence of the FER1 and that of other amino acid sequence of the common ticks of the family Ixodidae.The CDs region of the FER1 gene encodes 172 amino acids,and the theoretical isoelectric point(p I)is 5.04.The protein exists in the form of 24-mer with a hollow inside,and the antigen sites of a single subunit located in the amino acids of aa8-aa12 and aa80-aa93,which are located on the surface of the 24-mer model,showing antigenicity.Amino acid linear fragment predicted that FER1 did not show anaphylaxis.The FER1 was cloned into the prokaryotic expression vector,resulting a prokaryotic expression vector p ET-28 a-FER1.The recombinant protein was purified by His trap affinity chromatography.The size of the protein was 25 ku.The reactogenicity of r FER1 was detected by western blot.In this study,the sequence homology of FER1 protein and the phylogenetic relationship of FER1 gene were analyzed,and the spatial structure and antigenicity of the protein were predicted.Through the expression of the recombinant protein and serological experiments,it was inferred that FER1 may exist in the salivary gland of ticks,indicating that the protein has a certain candidate antigen potential for anti-tick vaccine.