多房棘球绦虫角化蛋白的基因克隆、表达及免疫反应性分析  

Cloning,expression,and analysis of the immunoreactivity of the cornifelin gene of Echinococcus multilocularis

作　　者：廖鹏[1,2] 李想 谭青青[1,2] 杨新琦 曹纹侨 张静 叶彬 LIAO Peng;LI Xiang;TAN Qing-qing;YANG Xin-qi;CAO Wen-qiao;ZHANG Jing;YE Bin(Department of Pathogen Biology,Chongqing Medical University,Chongqing 400016,China;Center for Molecular Medicine and Tumor Research,Chongqing Medical University)

机构地区：[1]重庆医科大学病原生物学教研室,重庆400016 [2]重庆医科大学分子医学与肿瘤研究中心

出　　处：《中国病原生物学杂志》2021年第10期1187-1192,1197,共7页Journal of Pathogen Biology

摘　　要：目的克隆表达多房棘球绦虫角化蛋白(EmCNFN)基因,并分析其免疫反应性。方法以细粒棘球绦虫CNFN氨基酸序列为探针,通过电子克隆方法获得EmCNFN基因cDNA全长。设计EmCNFN特异性引物,PCR扩增EmCNFN基因,并进行菌落PCR和测序验证。构建pET32a-EmCNFN原核表达载体,筛选IPTG最适诱导浓度和最适诱导时间进行诱导表达,制备EmCNFN重组蛋白。通过Ni柱层析获取高纯度的EmCNFN重组蛋白。分别用His标签抗体、泡球蚴病患者血清、健康人血清和泡球蚴感染SD大鼠血清、健康SD大鼠血清作一抗,Western blot分析EmCNFN重组蛋白免疫反应性。采用生物信息学软件分析EmCNFN蛋白潜在B、T细胞表位。结果成功获得EmCNFN基因cDNA全长,PCR试验和测序验证其序列正确。优化的IPTG最佳诱导浓度为0.5 mmol/L,最佳诱导时间为8 h。原核表达后通过Ni柱层析,获得了高浓度高纯度的40 ku EmCNFN重组蛋白。Western blot显示,EmCNFN重组蛋白可与His标签抗体、泡球蚴病患者血清和泡球蚴感染SD大鼠血清发生特异性反应。生物信息学软件分析发现,EmCNFN蛋白含有4个潜在B细胞表位,8个潜在T细胞表位。结论成功获得EmCNFN基因cDNA序列,克隆表达、纯化了EmCNFN重组蛋白,该蛋白含有B、T细胞表位,具有免疫反应性,为进一步研究其功能奠定了基础。Objective To clone and express the cornifelin(EmCNFN) gene of Echinococcus multilocularis and to analyze its immunoreactivity. Methods The full-length cDNA sequence of the EmCNFN gene was obtained via electronic cloning using the CNFN amino acid sequence of E. granulosus as a probe. Specific primers were designed based on the sequence of the EmCNFN gene, and the gene was amplified using PCR with cDNA from E. multilocularis metacestodes as a template. The prokaryotic expression vector pET32 a-EmCNFN was constructed and transformed into E.coli BL21, and the optimal concentration of IPTG and the optimal timing for induction of expression were screened for in order to prepare the recombinant protein EmCNFN. Highly pure recombinant EmCNFN protein was obtained using Ni column chromatography. The immunoreactivity of the recombinant protein EmCNFN was analyzed with Western blotting using His tag antibodies, sera from patients with alveolar echinococcosis(AE), healthy human sera, and sera from SD rats infected with E. multilocularis and healthy SD rats as primary antibodies. Bioinformatic software was used to analyze the potential B-and T-cell epitopes of the EmCNFN protein. Results The full-length cDNA sequence of the EmCNFN gene was successfully obtained and was verified as correct using amplification with PCR and sequencing. The optimal concentration of IPTG for induction of expression was optimized to 0.5 mmol/L, and the optimal timing for induction of expression was determined to be 8 h. After prokaryotic expression by E.coli BL21, expression of about 40 ku of the recombinant EmCNFN protein was successfully induced, and then protein at a high concentration and a high level of purity was obtained using Ni column chromatography. Western blotting indicated that the recombinant EmCNFN protein specifically reacted with His tag antibodies, sera from patients with AE, and sera from SD rats infected with E. multilocularis but it did not react with normal sera from humans and SD rats. Bioinformatic software indicated that the

关 键 词：泡球蚴病 角化蛋白 生物信息学 原核表达 表位 

分 类 号：R383.3[医药卫生—医学寄生虫学]