家蚕过敏原Profilin的表达、纯化、免疫学鉴定及其生物信息学分析

Expression,purification and bioimformatics analysis of allergen protein Profilin from silkworm( Bombyx mori)

目的:克隆表达家蚕(Bombyx mori)Profilin蛋白,鉴定其免疫原性并进行B细胞抗原表位预测和构建分子进化树。方法:从NCBI上获取家蚕Profilin蛋白的基因序列,合成该基因并构建到pet-28a表达载体上,重组质粒转化至E.coli BL21,IPTG诱导基因表达,通过亲和层析获取高纯度蛋白;用Western blot方法对重组蛋白进行过敏原性鉴定;通过DNAStar软件分析其潜在的B细胞抗原表位;应用MEGA5.05进行序列比对,并构建分子进化树。结果:成功表达出重组蛋白,且重组蛋白与家蚕过敏患者血清有一定的Ig E结合。结论:成功克隆表达出具有免疫原性的Profilin蛋白,并成功预测出其B细胞抗原表位和构建出分子进化树。

Objective： To obtain recombinant Profilin of silkworm,identify its immunogenicity,predict its B cell epitopes and construct the evolutionary trees. Methods： The nucleotide sequence of Profilin was acquired from NCBI,synthesized it and cloned it into p ET-28 vector. Then,the recombinant plasimids were transformed to E. coli BL21. After induced by IPTG,recombinant protein was purified by Affinity chromatography. Furtherly,its allergenicity was identified by Western blot,the potential B cell epitopes was analyzed through DNAStar and build the evolutionary trees by MEGA5. 05. Results： The recombinant protein of Profilin was successfully expressed and purified by affinity chromatography. Besides,the protein contains a high Ig E-binding activity with Ig E existing in serum of patients allergic to silkworm. Conclusion： The recombinant proflilin has Ig E-binding activity,and it is meaningful for fundamental research and specific diagnosis studies of allergic diseases caused by silkworm.