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目的 纯化重组日本血吸虫特异性IgE抗体相关蛋白和鉴定其免疫原性。 方法 大量表达Sj43B/pGEX 6p 1 /BL2 1重组克隆菌 ,超声粉碎后离心获得融合表达的重组蛋白包涵体。经TNMFX缓冲液分步洗涤后 ,将包涵体溶解液经FPLC分离 ,获得重组融合蛋白组分 ,再经巯基二硫键转换复性后 ,用于免疫小鼠获得抗血清 ,分别用dot ELISA法和Westernblotting法对融合蛋白引起的特异性血清抗体同型反应进行鉴定。 结果 分步洗涤可有效去除重组蛋白包涵体沉淀中混杂的多数杂蛋白成分 ,FPLC分离可获得高纯度重组蛋白。用复性后的重组融合蛋白免疫小鼠 ,其中目的蛋白可引起特异性IgE抗体反应 ,而担体蛋白 2 6kDaGST不引起特异性IgE应答 ,可引起特异性IgG抗体反应 ,目的蛋白则否。 结论 重组质粒Sj43B/pGEX 6p 1表达的融合蛋白 ,其目的蛋白部分免疫小鼠可产生特异性IgE抗体。
Objective To purify recombinant Schistosoma japonicum specific IgE antibody - related protein and identify its immunogenicity. Methods Sj43B / pGEX 6p 1 / BL2 1 recombinant clones were overexpressed and were pulsed with ultrasound and centrifuged to obtain fusion protein inclusion bodies. After stepwise washing with TNMFX buffer, the inclusion body lysates were separated by FPLC to obtain recombinant fusion protein components, which were then refolded by mercapto-disulfide conversion and then used to immunize mice to obtain antisera. The dot ELISA assay Western blotting was used to identify the specific serum antibody homotypes induced by the fusion protein. The results of step-by-step washing can effectively remove most of the hybrid protein components in the recombinant protein inclusion bodies precipitation, FPLC separation can be obtained high purity recombinant protein. Mice were immunized with the refolded recombinant fusion protein, in which the target protein elicited a specific IgE antibody response, whereas the carrier protein 26 kDa GST did not elicit a specific IgE response and elicited specific IgG antibody responses without the target protein. Conclusion The fusion protein expressed by recombinant plasmid Sj43B / pGEX 6p 1 can produce specific IgE antibodies by immunizing mice with the target protein.