中国寄生虫学与寄生虫病杂志

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日本血吸虫重组抗原rSj26的磁分离酶联免疫分析的建立及其在低感染度血清抗体检测中的应用

余琴1*,朱艳红2,关飞3,杨海2   

  1. 1 武汉血液中心检验科,武汉 430030;2 华中科技大学生命科学院,武汉 430074;3 华中科技大学同济医学院寄生虫学教研室,武汉 430030
  • 出版日期:2016-08-30 发布日期:2016-11-07
  • 基金资助:

    Supported by the National Natural Science Foundation of China(No. 81402640),Natural Science Fund of Hubei Province (No. 2014CFB406),Research Project of Health and Family Planning Commission of Wuhan Municipality(No. WX15B23),and Young and Middle-Aged Medical Personnel Training Project of Wuhan Municipality(No. 2014-77).

Establishment of Magnetic Affinity Enzyme Linked Immunoassay Based on Schistosoma japonicum Recombinant Antigen Sj26 and Its Application in Detection of Serum Antibody with Low Intensity of Infection

YU Qin1*, ZHU Yan-hong2, GUAN Fei3, YANG Hai2   

  1. 1 Department of Clinical Laboratory, Wuhan Blood Center, Wuhan 430030, China;2 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China;3 Department of Parasitology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
  • Online:2016-08-30 Published:2016-11-07

摘要:

目的 建立基于重组日本血吸虫抗原Sj26(rSj26)的磁分离酶联免疫分析(rSj26-MEIA),并将其用于检测低感染度的日本血吸虫病患者的血清抗体。 方法 将重组质粒pET28a-Sj26转化至大肠埃希菌(Escherichia coli)BL21,用0.6 mmol/L 异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,表达产物经镍柱亲和层析法纯化后,二喹啉甲酸(BCA)法测定蛋白含量,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹(Western blotting)检测分析rSj26。将纯化后的rSj26与磁珠偶联(100 μg抗原/mg磁珠),优化反应条件,建立rSj26-MEIA方法。用rSj26-MEIA和ELISA分别检测58份低密度感染的日本血吸虫病患者血清、30份非血吸虫病流行区阴性血清和6份并殖吸虫病患者血清,并比较两者的检测结果。 结果 纯化后的重组蛋白含量测定结果显示,rSj26浓度为2.5 mg/ml。SDS-PAGE结果显示,rSj26相对分子质量约27 000,主要以可溶性的形式表达。Western blotting结果显示,rSj26可被感染日本血吸虫的兔血清和小鼠血清特异识别。rSj26-MEIA优化反应条件结果显示,采用rSj26-磁珠0.2 mg(含rSj26 10 μg)、血清稀释度为1 ∶ 100时,阳性血清平均吸光度(A550值)/阴性血清平均A550值(P/N)最大,为3.97。rSj26-MEIA和rSj26-ELISA检测低密度感染日本血吸虫病患者血清的结果显示,两者的阳性检出率均为24.14%(14/58),两者P/N分别为3.61和2.56;相关分析结果表明,两者检测的抗体A550值间存在正相关关系(r=0.658,P<0.01)。rSj26-MEIA和ELISA检测6例并殖吸虫病患者血清和30例非血吸虫病流行区阴性血清,均未出现阳性反应。 结论 rSj26-MEIA可作为检测低密度感染日本血吸虫血清抗体的一种新技术。

关键词: 日本血吸虫, 重组抗原Sj26, 抗体, 磁分离酶联免疫法, ELISA

Abstract:

Objective To develop the magnetic affinity enzyme-linked immunoassay(MEIA) using recombinant glutathione-S-transferase of Schistosoma japonicum with a relative molecular weight of 26 000(rSj26-MEIA) for antibody detection under low intensity infection. Methods The recombinant plasmid pET28a-Sj26 was transformed into Escherichia coli strain BL21, and 0.6 mmol/L isopropyl β-D-1-thiogalactopyranoside (IPTG) was used to induce its expression. The expression products were purified by Ni2+ (nickel sulfate) affinity chromatography, SDS-PAGE and Western blotting were performed to examine the expression of rSj26. The purified rSj26 was coupled to magnetic beads as capture antigen and the reaction conditions were optimized to establish the rSj26-MEIA method. The method was then used to analyze 58 serum samples from patients with low-intensity S. japonicum infection, 30 serum samples from non-endemic areas as a negative control, and 6 serum samples from patients with paragonimus infection. Results were compared with those obtained with ELISA. Results The concentration of purified rSj26 was 2.5 mg/ml. The rSj26 had a relative molecular weight of 27 000 and was expressed mainly in the soluble form as revealed by SDS-PAGE. It could be recognized by rabbit and murine sera infected with S. japonicum as shown by Western blotting. Optimization of rSj26-MEIA revealed that use of 0.2 mg magnetic beads loaded with 10 μg rSj26 and serum sample dilution at 1 ∶ 100 yielded the highest ratio of the mean A550 of positive serum to the mean A550 of negative sample(P/N)(3.97). For serum samples from patients with low-intensity S. japonicum infection, rSj26-MEIA and rSj26-ELISA both resulted in a positive detection rate of 24.14%(14/58), and P/N values of 3.61 and 2.56. In addition, Pearson′s correlation analysis revealed positive correlation between A550 values detected by rSj26-MEIA and by rSj26-ELISA(r=0.658, P<0.01). Further, no positive reaction was found in the 6 serum samples from patients with paragonimus infection and in the 30 serum samples from non-endemic areas, either by rSj26-MEIA or by rSj26-ELISA. Conclusion rSj26-MEIA may be used as a new technique for detection of serum antibody against S. japonicum infection with low intensity.
  

Key words: Schistosoma japonicum, rSj26, Antibody, Magnetic affinity enzyme-linked immunoassay, ELISA