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目的对家蚕过敏原tropomyosin基因进行克隆表达,鉴定其免疫学活性。方法本研究提取家蚕总RNA,采用RT-PCR方法有效地扩增出tropomyosin片段,其长度为858bp,编码286个氨基酸。通过连入载体PET28a在大肠杆菌EscherichiacoliBL21(DE3)菌株中通过IPTG诱导得到重组家蚕原肌球蛋白。重组家蚕原肌球蛋白经过6His-tag蛋白纯化系统分离、纯化。经Westernblot检测该重组蛋白与家蚕过敏患者血清中IgE的反应性。结果19个病人血清反应中,有5个呈阳性,阳性率为26%。结论成功克隆表达了家蚕tropomyosin基因,经免疫学鉴定发现其具有过敏原性。
Objective To clone and express tropomyosin gene of silkworm (Bombyx mori) and identify its immunological activity. Methods The total RNA of silkworm was extracted from this study. The tropomyosin fragment was amplified by RT-PCR and its length was 858bp and encoded 286 amino acids. Recombinant silkworm tropomyosin was induced by IPTG in the Escherichia coli BL21 (DE3) strain of Escherichia coli by ligating into the vector PET28a. The recombinant silkworm tropomyosin was separated and purified by 6His-tag protein purification system. Western blot was used to detect the reactivity of the recombinant protein with IgE in serum of silkworm allergy patients. Results Nineteen of the 19 patients had positive serological responses, with a positive rate of 26%. Conclusion The silkworm tropomyosin gene was successfully cloned and expressed, and it was found to be allergenic by immunological identification.