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酵母表达的重组戊型肝炎病毒结构区ORF2蛋白的抗原性鉴定 被引量:2

Antigencity identification of recombinant hepatitis E virus ORF2 protein expressed in Pichia pastoris
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摘要 目的对应用巴氏毕赤酵母表达系统制备的重组戊型肝炎病毒(HEV)结构区ORF2蛋白的抗原性进行鉴定.方法以原核细胞表达的重组HEV ORF2抗原作为对照,应用间接ELISA方法鉴定重组HEV ORF2蛋白在HEV IgM和IgG抗体检测中的特异性和敏感性;并测定不同保存时间对抗原稳定性的影响.同时,比较了两种重组抗原与国外5株HEV ORF2单克隆抗体的反应特性.结果应用酵母抗原可检测到HEV抗体的最低包被量为12.5 ng/ml,可检测到HEV IgM、IgG抗体的血清最大稀释度皆为1:5 120.重组蛋白与其他类型肝炎患者血清无交叉反应,37℃加速试验证实重组蛋白4℃保存12个月可保持良好的抗原性.各株单克隆抗体与酵母表达的HEV ORF2蛋白有更好的反应性,其中4B2、2E2与酵母表达的HEV ORF2蛋白的反应性比与原核表达抗原的反应性分别高出125倍和25倍.结论应用巴氏毕赤酵母表达系统制备的重组HEV ORF2蛋白可能包含了更为广泛的构象依赖性抗原表位,具有良好抗原性、特异性和稳定性.提示其可作为开发戊型肝炎诊断试剂的一个独具优势的候选抗原. Objective To determine the antigencity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P pastoris) Methods By using the rHEV ORF2 protein from E coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested Results The minimum concentration of coated antigen with which HEV IgG could be detected was 12 5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1∶5 120 No cross reaction was found with sera from patients with any other types of hepatitis The 37℃ acceleration test showed that the rORF2 was highly stable within 12 months at 4℃ The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P pastoris , especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E Coli Conclusion There may be more extensive conformational epitopes in the rHEV ORF2 from P pastoris The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E
作者 佟玉品 毕胜利 鲁健 江永珍 詹美云 TONG Yu pin *, BI Sheng li, LU Jian, JIANG Yong zhen, ZHAN Mei yun *The No.261 Hospital of PLA, Beijing,100094, China
出处 《中华实验和临床病毒学杂志》 CAS CSCD 北大核心 2003年第3期 258-261,共4页 Chinese Journal of Experimental and Clinical Virology
关键词 酵母 重组戊型肝炎病毒 ORF2蛋白 抗原 ELISA 戊型肝炎 Hepatitis E virus Open reading frames Recombinant proteins Antigens Pichia
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