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目的 克隆查菲埃立克体 12 0kDa膜表面抗原蛋白基因 ,获得纯化的重组蛋白。方法 根据查菲埃立克体(91HE17) 12 0kDa抗原蛋白基因序列设计特异性引物 ,PCR扩增查菲埃立克体 12 0kDa抗原蛋白的基因片段 ;用限制性内切酶酶切PCR扩增产物后与 pUC18载体相连接 ,经酶切和序列分析证实后 ,将目标片段定向插入原核表达载体pProEXHTB中构建pProEXHTB/ p12 0重组质粒 ;将重组质粒转化E coliDH5α ,并使转化子在IPTG的诱导下进行蛋白质表达 ;运用亲和层析和电洗脱法对重组蛋白进行纯化。结果 克隆到一大小为 10 80bp的查菲埃立克体 12 0kDa抗原蛋白的基因片段 ,用该基因与表达质粒连接 ,成功构建了重组表达质粒 ;SDS -PAGE分析显示 :在IPTG诱导下 ,重组表达质粒转化的大肠杆菌产生一分子量为 4 7kDa的目标蛋白 ,免疫印迹证明该重组蛋白能与查菲埃立克体免疫血清发生反应。用纯化的重组蛋白作免疫斑点分析 ,76份被检血清中有 2份为可疑阳性 ,但经IFA复核为阴性。结论 查菲埃立克体 12 0kDa重组抗原蛋白具有免疫反应活性 ,为以重组蛋白为基础的人单核细胞埃立克体病的血清学诊断试剂盒制备和疫苗的研制奠定了基础。
Objective To clone the 120 kDa membrane surface antigen protein gene of Chaffy Ehrlichiae and obtain the purified recombinant protein. Methods The specific primers were designed according to the sequence of 12 0 kDa antigen of Chafeffy Erikson (91HE17), PCR was used to amplify the gene fragment of 120 kDa antigen of Chafira Ehrlichiae by restriction endonuclease digestion After the product was ligated to the pUC18 vector, the fragment was inserted into the prokaryotic expression vector pProEXHTB to construct the recombinant plasmid pProEXHTB / p12 0 after confirmed by restriction analysis and sequence analysis. The recombinant plasmid was transformed into E. coli DH5α and the transformants were induced in IPTG Under the protein expression; using affinity chromatography and electroelution method of recombinant protein purification. Results A gene fragment of 120 kDa antigen of Chaffer Ehrlichia was cloned into a size of 10 80bp. The recombinant plasmid was successfully constructed by ligating the gene with the expression plasmid. SDS-PAGE analysis showed that under the induction of IPTG, Escherichia coli transformed with the expression plasmid produced a target protein with a molecular weight of 47 kDa, and immunoblotting confirmed that the recombinant protein reacted with the immunofluorescence of Chafiri Ehrlichiae. Using purified recombinant protein for immunoblot analysis, 2 out of 76 sera tested were suspicious positive but negative after IFA review. Conclusion The 120 kDa recombinant antigen of Chaffer Ehrlichiae is immunoreactive and lays a foundation for the preparation of serological diagnosis kit for human monocytic ehrlichiosis and the development of vaccine based on the recombinant protein.