肝片吸虫SAP-2蛋白的基因克隆、表达及其诊断潜力初步评估

Cloning,Expression of SAP-2 Gene of Fasciola hepatica and Its Preliminary Assessment of Diagnostic Potential

【目的】为了研究肝片吸虫(Fh) SAP-2蛋白的反应原性及作为Fh血清学诊断抗原的潜力。【方法】本研究利用RT-PCR方法对Fh SAP-2基因进行扩增,将扩增产物克隆入p MD19-T载体后测序,对其进行分子特征分析;构建原核表达载体p ET-SAP-2,在E. coli BL21(DE3)中进行表达,通过SDS-PAGE和Western blot对表达的重组蛋白进行反应原性分析;以纯化的重组蛋白SAP-2(re SAP-2)为抗原,建立了检测Fh特异性抗体的间接ELISA,评估了SAP-2蛋白作为Fh血清学诊断抗原的潜力。【结果】Fh SAP-2基因全长306 bp,编码101个氨基酸。在线软件分析发现,SAP-2为疏水性、无跨膜区蛋白,信号肽位于第1～15个氨基酸之间。系统进化树分析表明,Fh SAP-2与曼氏裂体吸虫同源性最高,与其他寄生虫存在较大的差异。Western blot证实,re SAP-2蛋白可被Fh阳性血清特异性识别,有较强的反应原性。用re SAP-2蛋白建立了间接ELISA,对23份Fh阳性血清和10份阴性血清进行检测,结果证实re SAP-2蛋白有良好的特异性和敏感性。【结论】Fh SAP-2蛋白具有较强的反应原性,具有作为Fh血清学诊断抗原的潜力。

【Objective】The study aimed to investigate the reactivity of SAP-2 protein of Fasciola hepatica(Fh)and its potential as a serological diagnostic antigen for Fh.【Method】In this study,Fh SAP-2 gene was amplified by RT-PCR and then cloned it into pMD19-T vector and analyzed its molecular characteristics;The prokaryotic expression vector pET-SAP-2 was constructed and expressed in E.coli BL21(DE3),and analysis of the reactivity of the recombinant protein by SDS-PAGE and Western blot.The purified recombinant protein SAP-2(reSAP-2)was used as antigen to establish an indirect ELISA for detection of Fh specific antibodies.【Result】The Fh SAP-2 gene was 306 bp in length,encoding 101 amino acids.The online software analyzed that the SAP-2 is a hydrophobic and no transmembrane protein,and the signal peptide was located between one and fifteen amino acids.Phylogenetic tree analysis showed that Fh SAP-2 had the highest homology with Schistosoma mansoni,but there were significant differences with other parasites.Western blot confirmed that the reSAP-2 protein can be specifically recognized by the positive serum of Fh,which proved it had good reactivity.Indirect ELISA was established by reSAP-2 protein,and twenty three Fh positive sera and ten negative sera were detected and the results showed that the reSAP-2 protein had good specificity and sensitivity.【Conclusion】Fh SAP-2 protein has strong reactogenicity and has potential as a Fh serological diagnostic antigen.