查菲埃立克体重组120kDa抗原的初步应用研究

Preliminary study on the 120-kDa recombinant antigen protein of Ehrlichia chaffeensis

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 gene encoding 120-kDa major immunodominant outer membrane protein of E.chaffeensis.and purify the recombinant protein.Methods Specific primers used to amplify E.chaffeensis DNA were synthesized on the basis of P120 protein gene sequence of E.chaffeensis.PCR products were digested by restriction enzyme BamHⅠand EcoRⅠand ligated with pUC18 vector.After identification by restriction enzyme and DNA sequencing,then the targeted gene was cloned into pProEX HTb expression vector.E.coli DH5α was transformed with the recombinant plasmid pProEX HTb/P120.Expression of P120 gene in the DH5α was induced by IPTG.The electroelution and Ni-NTA affinity chromatography were used to purify the recombinant protein.The recombinant protein was used in dot immunoblotting for the detection of the serum of suspected patients.Questionable serums were reexamined by IFA.Results A 1080bp gene of P120 protein was cloned into pUC18 vector.E.coli DH5α transformed with the recombinant plasmid pProEX HTb /P120 was induced by IPTG to express a fusion protein with the expected molecular mass of 47kDa.Western Blot revealed that the recombinant protein could react with immued serum.Targeted protein was achieved by protein purification.2 of 76 sera showed positive reaction in immuno-dot blotting,however,they were negative in IFA.Conclusion P120 gene of E.Chaffeensis can be expressed in E.coli.Antibodies in animal's serum could react with the combinant E.chaffeensis 120-kDa protein.The work provides the basis for development of detection kits and other researches on E.chaffeensis.