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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(100μg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 10μg)、血清稀释度为1∶100时,阳性血清平均吸光度(A_550值)/阴性血清平均A_550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A_550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。
Objective To establish a magnetic separation enzyme-linked immunosorbent assay (rSj26-MEIA) based on the recombinant Schistosoma japonicum antigen Sj26 (rSj26) and use it for the detection of serum antibodies to schistosomiasis japonica patients with low infectivity. Methods The recombinant plasmid pET28a-Sj26 was transformed into Escherichia coli BL21 and induced with 0.6 mmol / L isopropyl-β-D-thiogalactoside (IPTG) After purification by chromatography, the content of protein was determined by bicinchoninic acid (BCA) assay, rSj26 was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The purified rSj26 was coupled with magnetic beads (100 μg antigen / mg magnetic beads), the reaction conditions were optimized and the rSj26-MEIA method was established. Fifty-eight sera of patients with low-density infection of Schistosoma japonicum, 30 sera of non-schistosomiasis endemic areas and 6 paragonimiasis were detected by rSj26-MEIA and ELISA, respectively. The results of the two tests were compared. Results The purified recombinant protein showed that the concentration of rSj26 was 2.5 mg / ml. SDS-PAGE showed that the relative molecular mass of rSj26 was about 27 000, which was mainly expressed in soluble form. Western blotting results showed that rSj26 could be specifically recognized by rabbit sera and mouse sera infected with Schistosoma japonicum. The optimal reaction conditions of rSj26-MEIA showed that the positive serum mean absorbance (A_550) / negative serum mean A_550 (P / N) was obtained with rSj26-magnetic beads 0.2 mg (rSj26 10 μg) at the serum dilution of 1: The maximum is 3.97. The results of rSj26-MEIA and rSj26-ELISA in sera of patients with low-density infection of schistosomiasis japonica showed that the positive rates of both were 24.14% (14/58), the P / N ratios were 3.61 and 2.56 respectively. Correlation analysis The results showed that there was a positive correlation between A_550 values of the two antibodies (r = 0.658, P <0.01). Serum samples from 6 patients with paragonimiasis and 30 negative samples from non-endemic areas of schistosomiasis were detected by rSj26-MEIA and ELISA. No positive reaction was observed. Conclusion rSj26-MEIA can be used as a new technique to detect serum antibody of Schistosoma japonicum infected with low density.