细粒棘球蚴中国大陆株亲肌肉抗原重组蛋白的表达、纯化及免疫学鉴定

目的对细粒棘球蚴中国大陆株亲肌肉基因(myophilin)的重组质粒进行原核表达、纯化,并初步鉴定重组蛋白的免疫学特性,为重组疫苗研制的后续工作提供依据。方法对已构建的基因工程菌株myophilin/pGEM-T/JM109进行酶切,获取目的基因。将目的基因亚克隆于表达载体pET28a构建基因工程菌株,筛选阳性表达菌株并进行诱导表达、经SDS-PAGE鉴定重组myophilin蛋白。以纯化的myophilin做抗原免疫小鼠,用Western blot、ELISA对该蛋白的免疫原性及免疫鼠血清抗体水平进行检测。结果成功构建含原核重组表达载体myophilin/PET28a/BL21,并纯化出浓度较高的重组蛋白;ELISA检测显示,用表达、纯化的重组蛋白免疫小鼠,诱导产生了一定水平的血清抗体;实验组血清与对照组血清抗体含量差异有统计学意义(P<0.05)。结论纯化后的重组蛋白myophilin具有较强的免疫原性,有望作为包虫病候选疫苗进一步研究。

Expression,purification and Immunogenicity Analysis on Myophilin Recombinant Protein of Echinococcus Granulosus (Chinese mainland strain)

Objective To express myophilin of Echinococcus granulosus(Eg myophilin Chinese mainland strain)recombinant plasmid prokaryotically,to purify and identify the immunogenicity of recombinant myophilin protein in order to provide more information to further development of vaccines for hydatid disease.Methods By digesting with BamHI and NotI on myophilin/pGEM-T/JM109,Eg myophilin gene was obtained and linked with expression vector pET28a.myophilin/pET28a/BL21 was constructed and recombinant protein was induced by IPTG.Expressed Eg myophilin was identified as inclusion bodies by SDS-PAGE.The recombinant protein was injected into mice after purifying by affinity chromatography.Western blot and ELISA were used to identify the immunogenicity of the recombinant protein.Results myophilin/pET28a/BL21 of Eg myophilin recombinant plasmid was constructed and higher concentration recombinant protein was purified.ELISA showed that myophilin-immunized mice could induce a certain level of antibody and Western blot analysis revealed that myophilin-immunized mice could induce a certain level of antibody and Western blot showed that cyctic fluid and protoscolex could be recognized by the sera from recombinant protein immunized mice.Conclusion Purified myophilin showed higher immunogenicity and could be a new vaccine candidate to echinococcus disease.