【摘要】 【目的】克隆、表达纯化出家蚕Bombyx mori蚕蛹过敏原蛋白CPH30(cuticular protein hypothetical 30 precursor),并对其进行免疫学鉴定及B细胞抗原表位预测。【方法】通过蛋白组学方法分析CPH30蛋白的潜在过敏原性,人工化学合成该蛋白基因,将基因连接到p ET-28a载体上,重组质粒转化至大肠杆菌Escherichia coli BL21,IPTG诱导基因表达。通过镍亲和层析获取高纯度蛋白,用Western blot方法对蛋白进行免疫学鉴定。通过DNAStar软件分析其潜在的B细胞表位。【结果】成功构建出CPH30基因的表达载体;表达纯化出CPH30蛋白,SDS-PAGE电泳结果显示分子量为25 k Da左右;Western blot结果显示,CPH30蛋白与家蚕过敏患者血清具有Ig E结合性,证明CPH30具有免疫原性;利用DNAStar软件成功分析出氨基酸序列第57-69和150-158位为潜在的B细胞表位。【结论】成功克隆表达出CPH30蛋白,并成功鉴定出其免疫原性,为家蚕蚕蛹过敏反应性疾病的特异性诊断和疫苗治疗奠定理论基础。更多还原

【Abstract】 【Aim】To obtain the recombinant allergen CPH30 in pupae of the silkworm,Bombyx mori,and to detect its immunogenicity. 【Methods】The potential allergenicity of CPH30 protein was analyzed with proteomic methods. The CPH30 gene was synthesized by chemical method and introduced into p ET-28 a vector. The expression of the recombinant CPH30 was induced by IPTG. The allergenic activity of CPH30 protein was detected by Western blot. The potential B cell epitopes were analyzed using DNAStar. 【Results】After induction with IPTG,CPH30 protein was largely expressed in Escherichia coli Rosetta and purified by affinity chromatography. The molecular weight of the recombinant CPH30 protein is 25 k Da. The CPH30 protein show a high Ig E binding activity with the serum from silkworm-allergic patients,indicating that the recombinant CPH30 protein has immunogenicity. Prediction result using DNAStar software showed that the potential B cell epitopes of CPH30 are located between the 57th- 69 th and 150th- 158 th amino acids.【Conclusion】The purified CPH30 has high purity and immunogenicity.This study lays a foundation for the specific diagnosis,vaccine treatment and further experimental studies of allergic diseases caused by silkworm pupae.更多还原