查菲埃立克体120kDa抗原蛋白基因的克隆与表达

CLONGING AND EXPRESSION OF THE GENE ENCODING 120-kDa ANTIGEN PROTEIN OF EHRLICHIA CHAFFEENSIS

目的 克隆查菲埃立克体 12 0kDa膜表面免疫决定性蛋白基因 ,并使之在原核表达系统中表达。方法 查菲埃立克体分离株 (91HE17)感染DH82细胞 40天后收集DH82细胞。根据查菲埃立克体P12 0蛋白基因序列设计特异性引物 ,套式PCR扩增查菲埃立克体P12 0蛋白基因部分片段 ,将PCR扩增产物用BamHⅠ和EcoRⅠ限制性内切酶双酶切后与 pUC18载体连接 ,在获得阳性克隆子后用限制性内切酶将克隆片段切下 ,定向插入原核表达载体pProEXHTb构建 pProEXHTb/P12 0重组质粒。将重组质粒转化E .coliDH5α ,并使之在IPTG的诱导下进行蛋白质表达。结果 裁短成 10 80bp的查菲埃立克体P12 0蛋白基因克隆到 pUC18载体 ,用IPTG诱导 pProEXHTb/P12 0重组质粒转化的E .coliDH5α ,表达一分子量大小约为 47kDa的融合蛋白。结论 查菲埃立克体 12 0kDa抗原蛋白基因能在原核表达系统中表达 ,为诊断试剂盒的研制及其它研究工作提供了基础。

Aim Cloning and expression of the gene encoding 120-kDa immunodominant major outer membrane protein of E.chaffeensis. Methods DH82 cells were inoculated with E.chaffeensis(91HE17) and collected after 40 days.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 EcoRⅠ,then was cloned into pUC18 vector and pProEX HTb expression vector.Escherichia coli DH5αwas transformed with the recombinant plasmid HTB/P120.Expression of P120 gene in the DH5αwas induced by IPTG.Result A 1080bp gene of P120 protein was cloned into pUC18 vector.E.coli DH5αtransformed with the recombinant plasmid pProEX HTb /P120 was induced by IPTG to express a fusion protein with the expected molecular mass of 47kDa.Conclusion P120 gene of E.Chaffeensis can be expressed in E.coli.The work provides the basis for development of detection kits and other researches on E.chaffeensis.