摘要
目的对细粒棘球蚴(中国大陆株)线粒体苹果酸脱氢酶(Eg mMDH)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。方法对已构建的基因工程菌株mMDH/pGEM-T/JM109进行酶切,获取目的基因。将目的基因重组到pET28a表达载体上,筛选阳性表达菌株并进行诱导表达,经SDS-PAGE鉴定重组mMDH蛋白质为包涵体后,超声破碎细胞,尿素溶解包涵体,镍柱亲和层析法纯化,获取重组蛋白。以纯化的mMDH作抗原免疫小鼠,用Western blot和ELISA方法检测重组蛋白的免疫原性及免疫鼠血清抗体水平。结果成功构建原核重组表达载体mMDH/pET28a/BL21,并纯化出浓度较高的重组蛋白。ELISA结果显示,重组抗原免疫小鼠诱导产生了一定水平的血清抗体;Western blot显示,原头蚴、囊液等天然抗原能被重组抗原免疫的小鼠血清识别。结论纯化后的重组蛋白mMDH具有较强的免疫原性,有望作为包虫病候选疫苗,值得进一步深入研究。
Objective To express mitochondrial malate dehydrogenase of Echinococcus granulosus （Eg mMDH Chinese mainland strain）recombinant plasmid prokaryotically, to purify and identify the immunogenicity of recombinant mMDH protein. Methods By digesting with BamH I and Xhol I on mMDH/pGEM-T/JM109, Eg mMDH gene was obtained and linked with expression vector pET28a, mMDH/pET28a/BL21 was constructed and recombinant protein was induced by IPTG. Expressed Eg mMDH was identified as inclusion bodies by SDS-PAGE and ultrasonicated and urea were used to dissolve cell. 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 mMDH/pET28a/BL21 of Eg mMDH recombinant plasmid was constructed and higher concentration recombinant protein was purified. ELISA revealed that mMDH-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 mMDH showed higher immunogenicity and may be a new vaccine candidate to echinococcus disease.
出处
《中国病原生物学杂志》
CSCD
2006年第4期249-252,共4页
Journal of Pathogen Biology
基金
国家自然科学基金项目(No.30260105)
宁夏自然科学基金项目(No.NZ0540)
关键词
细粒棘球蚴
线粒体苹果酸脱氢酶(mMDH)
蛋白纯化
免疫鉴定
Echinococcus granulosus
mitochondrial malate dehydrogenase
protein purification
immunological identification
