人源α-烯醇化酶B细胞表位鉴定及其与牙龈卟啉单胞菌烯醇化酶的交叉反应预测

Identification of antigenic epitope of human α-enolase and cross-reactivity with porphyromonas gingivalis enolase

目的:预测鉴定人源α-烯醇化酶蛋白的B细胞抗原表位,探讨人源α-烯醇化酶与牙龈卟啉单胞菌烯醇化酶之间的交叉反应性。方法:以人源α-烯醇化酶蛋白序列为基础,综合应用多个软件预测B细胞线性表位;再以人源α-烯醇化酶的蛋白晶体结构为基础,采用4个生物信息学软件综合预测B细胞构象表位;利用原核表达系统对人源α-烯醇化酶蛋白进行表达、亲和层析法进行,纯化抗原免疫BALB/c小鼠,化学合成预测的线性表位片段检测抗体反应,确定人源α-烯醇化酶蛋白B细胞表位。利用Clustal X进行蛋白质序列比对和Swiss-Model进行同源模建,研究人源α-烯醇化酶和牙龈卟啉单胞菌烯醇化酶的交叉反应性。结果:人源α-烯醇化酶线性B细胞表位优势区域为9-25aa,28-43aa,70-85aa,163-178aa,229-244aa,280-295aa;构象性B细胞表位优势区域为:1-4/24-30/77-86/124aa,9-16/51-59aa,250-254/262-268/271-274aa;合成的线性表位片段中9-25aa免疫应答反应最强烈,其次是70-85aa,再次为28-43aa,163-178aa,229-244aa,280-295aa;牙龈卟啉单胞菌烯醇化酶和人源α-烯醇化酶氨基酸序列同源性高达50.11%,两者在线性B细胞表位优势区域的9-24aa和32-42aa序列高度相似;空间构象比对结果显示牙龈卟啉单胞菌烯醇化酶和人源α-烯醇化酶之间的RMSD值为1.055,且两者在3个构象性B细胞表位优势区域的空间构象高度相似,推测二者可能在上述区域发生交叉反应。结论:预测并鉴定了人源α-烯醇化酶蛋白的B细胞抗原表位,比较其与牙龈卟啉单胞菌的烯醇化酶可能发生交叉反应的位点,为研究人源α-烯醇化酶蛋白的自身抗原性和类风湿性关节炎发病机制提供了理论基础。

Objective： To predict B cell antigentic epitopes of human α-enolase and analyse the cross-reactivity between humanα-enolase and porphyromonas gingivalis enolase. Methods： Based on the sequence of human α-enolase,used six software to predict the B cell liner epitopes. Then based on the crystal structure,used four software to predict the conformational B cell epitopes. The human α-enolase was expressed by prokaryotic expression system and purified by affinity chromatography. Used purified human alpha-enolase as antigen to immune BALB / c mice,then detect the B cell immune response by the synthetised linear fragments. Used Clustal X to do the sequence alignment and Swiss-Model to do homology modeling,study the cross reactivity between human α-enolase and porphyromonas gingivalis enolase. Results： Human α-enolase potential linear B cell epitope regions were 9-25 aa,28-43 aa,70-85 aa,163-178 aa,229-244 aa,280-295 aa,conformational B cell epitopes were 1-4 /24-30 /77-86 /124 aa,9-16 /51-59 aa,250-254 /262-268 /271-274 aa. In the synthetic linear epitopes,9-25 aa had the most intense immune response,followed by was 70-85 aa,and then were 28-43 aa,163-178 aa,229-244 aa,280-295 aa. Sequence blast results showed that the homology between porphyromonas gingivalis enolase and human α-enolase was 50. 11%,among the predicted linear epitope,9-24 aa and 32-42 aa were highly similar. Conformation blast results showed that the RMSD value between them was 1. 055 ,the conformation in 1-4 /24-30 /77-86 /124 aa,9-16 /51-59 aa,250-254 /262-268 /271-274 aa region are highly similar,suggesting that the two may cross-react in the above areas. Conclusion： In this study,the potential B cell antigentic epitopes have been predicted and tested the by sythentic peptide,the cross-reactivity sequences between human α-enolase and porphyromonas gingivalis enolase have been study by sequence alignment and homology modeling. The results are helpful for the study of human α-enolase protein autoantigens and the pathogenesis of rheumatoid arthritis.