日本血吸虫核糖体蛋白SjRibosomal_L18a及其B细胞表位的筛选和评价

中国寄生虫学与寄生虫病杂志 ›› 2013, Vol. 31 ›› Issue (4): 5-270-274.

日本血吸虫核糖体蛋白SjRibosomal_L18a及其B细胞表位的筛选和评价

魏刚刚1，徐斌2，鞠川2，胡薇1,2 *

1 华东理工大学生物反应器工程国家重点实验室，上海200237；2 中国疾病预防控制中心寄生虫病预防控制所，卫生部寄生虫病原与媒介生物学重点实验室，世界卫生组织疟疾、血吸虫病和丝虫病合作中心，上海200025

出版日期:2013-08-30 发布日期:2013-12-27

Screening and Evaluation of Schistosoma japonicum SjRibosomal_L18a Protein and its B Cell Epitopes

WEI Gang-gang1, XU Bin2, JU Chuan2, HU Wei1,2 *

1 State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China; 2 National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention; Key Laboratory of Parasite and Vector Biology, MOH; WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China

Online:2013-08-30 Published:2013-12-27

摘要/Abstract

摘要： 目的筛选获得日本血吸虫（Schistosoma japonicum）核糖体蛋白（SjRibosomal_L18a）编码基因，预测其B细胞表位。克隆、表达重组蛋白SjRibosomal_L18a，并比较分析其与合成的B细胞表位的潜在诊断价值。方法利用B细胞表位预测软件筛选日本血吸虫蛋白质序列，获取函数打分值较高且抗原表位片段多的免疫原性蛋白，合成B细胞表位片段。生物信息学方法分析免疫原性蛋白基因及其编码蛋白的相对分子质量（Mr）、等电点、亲水性、信号肽、跨膜区和功能结构域等理化性质。制备日本血吸虫各虫期阶段总RNA，逆转录PCR（RT-PCR）扩增目的基因，分析各虫期转录本丰度。将扩增产物克隆至原核表达载体pET-28a中，重组质粒转化至大肠埃希菌（E. coli）BL21（DE3）菌株，异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达，组氨酸标记亲和层析法纯化重组蛋白。ELISA法比较分析重组蛋白和合成的B细胞表位的潜在诊断价值。结果预测软件筛选出免疫原性核糖体蛋白SjRibosomal_L18a以及2个B细胞表位（P1和P2）。SjRibosomal_L18a基因开放阅读框（ORF）由531个碱基组成，编码176个氨基酸，不含跨膜区和信号肽，为Mr 20 741，等电点为11.12。RT-PCR结果显示，各虫期均检测到SjRibosomal_L18a的转录本，且转录水平均较高。成功构建重组表达质粒SjRibosomal_L18a/pET-28a，诱导表达获得包涵体形式的重组蛋白，约为Mr 26 069。ELISA检测结果显示，重组蛋白、P1和P2检测15份慢性血吸虫病患者血清和15份健康人血清的敏感性和特异性分别为53.3%（8/15）和100%（15/15）、60%（9/15）和100%（15/15）、73.3%（11/15）和100%（15/15）。结论获得重组蛋白SjRibosomal_L18a及其免疫原性较高的表位片段P1、P2，P1和P2用于血清诊断的敏感性均高于重组蛋白。

关键词: , 日本血吸虫；B细胞表位；核糖体蛋白；虫期特异性

Abstract: Objective To screen a ribosomal protein（SjRibosomal_L18a） of Schistosoma japonicum and predict its B cell epitopes, and evaluate the potential diagnostic value of the recombinant protein and the synthetic B cell epitopes. Method S. japonicum protein sequences were screened and analyzed by using B-cell epitope prediction softwares. The immunogenic protein was selected based on the predicted score and the quantity of epitopes. The epitopes with higher score（P1 and P2） were synthesized. The relative molecular mass（Mr）, isoelectric point, grand average of hydropathicity, signal peptide, and transmembrane domain were predicted by bioinformatics tools. RT-PCR was used to analyze the transcription level of the different development stages. The encoding sequence was amplified by PCR, and cloned into pET28a vector. The recombinant plasmid was transformed into in E. coli BL21（DE3） cells and induced with IPTG. The recombinant SjRibosomal_L18a protein was purified with Ni-NTA resin. ELISA was used to evaluate the potential diagnostic value of the recombined protein and the synthetic B cell epitopes. Results SjRibosomal_L18a protein was obtained, its B cell epitopes and physicochemical properties were predicted. The open reading frame of SjRibosomal_L18a was composed of 531 bp, and encoded a 176-amino-acid protein with Mr 20 741, pI 11.12. RT-PCR result showed that this gene was transcribed at high level in each developmental stage. The recombinant plasmid SjRibosomal_L18a/pET-28a was constructed and the protein was expressed as inclusion bodies （Mr 26 069）. The sensitivity and specificity of recombined protein, P1 and P2 were 53.3% （8/15） and 100% （15/15）, 60% （9/15） and 100% （15/15）, 73.3% （11/15） and 100% （15/15）, respectively. Conclusion The recombinant protein（SjRibosomal_L18a） and its epitopes with higher immunogenicity are obtained. The sensitivity of the two epitopes （P1 and P2） was higher than that of SjRibosomal_L18a protein.

Key words: Schistosoma japonicum, B cell epitope, Ribosomal protein, Stage specificity

魏刚刚1，徐斌2，鞠川2，胡薇1,2 *. 日本血吸虫核糖体蛋白SjRibosomal_L18a及其B细胞表位的筛选和评价[J]. 中国寄生虫学与寄生虫病杂志, 2013, 31(4): 5-270-274.

WEI Gang-gang1, XU Bin2, JU Chuan2, HU Wei1,2 *. Screening and Evaluation of Schistosoma japonicum SjRibosomal_L18a Protein and its B Cell Epitopes[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2013, 31(4): 5-270-274.

[1]	沈继龙*. 弓形虫病的若干术语释义及实验室诊断的解读与处置策略[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(4): 13-382-386.
[2]	熊彦红, 郑彬*, 周晓农. 寄生虫病标准工作的SWOT分析[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(4): 17-338-341.
[3]	王兵丽*, 黄家福, 陈凡. 约氏疟原虫红内期体外培养的研究[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(4): 21-412-414.
[4]	邱竞帆，葛可，王勇*. 刚地弓形虫蛋白质组学研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(3): 11-214-218, 221.
[5]	姜晶1，2，赵权2，杨桂连1 *. 树突状细胞在蠕虫感染免疫应答中的作用[J]. 中国寄生虫学与寄生虫病杂志, 2015, 33(2): 16-147-150.
[6]	李娜1, 姜玉新2, 刁吉东1, 赵蓓蓓1, 李朝品1*. 粉尘螨Ⅲ类重组变应原对哮喘小鼠免疫治疗的效果[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(4): 7-280-284.
[7]	王东, 张媛媛. 隐孢子虫感染昆明小鼠后肠道病理变化及螺旋霉素治疗效果[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(3): 12-225-228.
[8]	王海龙1，殷丽天2，张铁娥1，关丽1，3，孟晓丽1，刘红丽1，殷国荣1 *. 刚地弓形虫棒状体蛋白17（ROP17）真核表达载体的构建、表达及其激酶活性分析[J]. 中国寄生虫学与寄生虫病杂志, 2014, 32(1): 6-29-33.
[9]	刘毅，奚卫. 流式细胞术在感染鼠疟原虫活红细胞检测中的应用[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 16-340-342.
[10]	马锐1,2，师志云1，王娅娜2，李宗吉2，孙俊峰2，赵巍1 *. 细粒棘球绦虫亲肌肉抗原重组蛋白诱导小鼠免疫应答的研究[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 4-357-360.
[11]	王小莉1，王媛媛1，方强1 *，薛玉芹1，沈继龙2. 外源性一氧化氮体外杀伤旋毛虫成虫的研究[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 7-374-377.
[12]	段梦娟1，段绩辉2，何国平1 *. 从疟疾控制阶段MIS系统的经验探讨建立消除阶段监控评估系统的模式[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 15-411-414.
[13]	郭绍华1, 周水森1 *, 黄芳1, 郑香1, 武松2, 周华云3, 卓玛央金4. 应用多重PCR法分析西藏察隅疟疾流行区按蚊吸血习性[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(2): 8-122-126.
[14]	温爱丹, 张皓冰*. 基于水解酶的抗寄生虫前药研究[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(2): 11-139-145.
[15]	贾林芝1, 马雅军2 *, 曹毅2, 钱锋2, 李翔宇2. 题库命题法下不同专业《医学寄生虫学》试卷结构差异成因分析[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(2): 14-160-163.