摘要
目的应用软件和在线网络分析EgG1Y162的氨基酸序列,了解其理化性质及二、三级结构特点;预测该抗原的T、B细胞表位,筛选优势表位并将其与热休克蛋白HSP70进行联合重组表达,验证该新型多表位疫苗刺激机体产生的免疫反应。方法通过NCBI检索获取EgG1Y162和HSP70的氨基酸序列,利用Protparam、NetPhos、TMHMM-2.0等在线程序分析EgG1Y162蛋白的分子质量、等电点、氨基酸组成、跨膜结构域、磷酸化位点,预测其二、三级结构及T、B细胞表位。结合预测结果优化确定最终的表位信息,将优化的优势表位信息与HSP70分子对接,构建重组HSP70-EgG1Y162,通过酶联免疫斑点试验(ELISPOT)检测对比分析HSP70蛋白、EgG1Y162和HSP70-EgG1Y162诱导产生的免疫反应强度。结果预测EgG1Y162蛋白由120 aa组成,HSP70由762 aa组成。在线分析EgG1Y162蛋白的二级结构中α-螺旋约占22.50%,延长链约占28.33%,β-转角约占6.67%,无规则卷曲约占42.50%。筛选EgG1Y162抗原的优势T、B细胞表位,然后以HSP70蛋白为载体与EgG1Y162抗原T-B细胞联合表位及连接蛋白Linker-GGGS构建HSP70-EgG1Y162多表位疫苗,免疫小鼠后取脾细胞进行ELISPOT检测,反应斑点的灰度值较对照组显著增高,即HSP70-EgG1Y162具有免疫原性。结论生物信息学方法分析预测细粒棘球绦虫EgG1Y162蛋白含有丰富的T、B细胞表位,筛选的优势表位与热休克蛋白HSP70融合后能够增强该新型包虫多表位抗原的免疫原性,ELISPOT证实以HSP70蛋白和EgG1Y162构建的HSP70-EgG1Y162多表位疫苗免疫原性增强,该设计方法有望成为设计优势疫苗的一种新方法,为包虫病的预防提供新思路。
Objective The purpose of this study is to analyze the amino acid sequence of the EgG1Y162 protein in order to understand its physicochemical properties and secondary and tertiary structure characteristics.Additionally,we aim to predict the T and B cell epitopes of this antigen,screen for dominant epitopes,and construct a recombinant vaccine by combining these epitopes with the heat shock protein HSP70.This will allow us to validate the immune response generated by this novel multi-epitope vaccine.Methods We obtained the amino acid sequences of EgG1Y162 and HSP70 from the NCBI database.We used online programs such as Protparam,NetPhos,and TMHMM-2.0 to analyze various properties of the EgG1Y162 protein,including its molecular weight,isoelectric point,amino acid composition,transmembrane domains,and phosphorylation sites.We also predicted the secondary and tertiary structures,as well as the T and B cell epitopes of EgG1Y162.Based on our predictions,we determined the optimal epitope information and docked the optimized dominant epitopes with the HSP70 molecule to construct a recombinant HSP70-EgG1Y162 vaccine.We compared and analyzed the immune response intensities induced by HSP70 protein,EgG1Y162,and HSP70-EgG1Y162 using the enzyme-linked immunospot assay(ELISPOT).Results We found that EgG1Y162 is composed of 120 amino acids,while HSP70 is composed of 762 amino acids.The online analysis of the secondary structure of EgG1Y162 protei showed that approximately 22.50%wereα-helices,approximately 28.33%were extended chains,approximately 6.67%wereβ-turns,and approximately 42.50%were irregular coils.We screened the dominant T and B cell epitopes of the EgG1Y162 antigen and used the HSP70 protein as a carrier to construct the HSP70-EgG1Y162 multi-epitope vaccine by combining the T-B cell epitopes of the EgG1Y162 antigen and a connecting protein linker-GGGS.ELISPOT analysis of spleen cells from immunized mice showed a significant increase in the grayscale value of the reaction spots compared to the control group,indicating
作者
张涛
郭久鹏
魏来
热甫克提·艾尼瓦尔
努尔阿米乃姆·库杜斯
于明凯
李玉娇
ZHANG Tao;GUO Jiupeng;WEI Lai;REFUKETI·ainiwaer;NUERAMINAIMU Kudusi;YU Mingkai;LI Yujiao(The First Clinical College,Xinjiang Medical University,Urumqi,Xinjiang 830011,China;Basic Medical College;First Affiliated Hospital)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第12期1424-1429,1433,共7页
Journal of Pathogen Biology
基金
国家自然科学基金项目(32160182)
新疆维吾尔自治区科学技术厅杰出青年项目(2022D01E69),新疆医科大学大学生创新训练计划项目(CX2021009)
新疆医科大学第一附属医院优秀青年人才培养专项。
