丙型肝炎病毒河北株E2蛋白胞外核心区的真核表达及应用  

Eukaryotic expression and application of HCV Hebei strain E2 extracellular core region

作　　者：叶传涛 边培育 翁代慧[2] 张会[2] 杨敬[2] 张颖[1] 雷迎峰[2] 贾战生[1] 

机构地区：[1]第四军医大学唐都医院传染科,陕西西安710038 [2]第四军医大学微生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2016年第6期746-749,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：十二五传染病重大专项(2013ZX10004609)

摘　　要：目的真核细胞表达丙型肝炎病毒(HCV)河北株E2蛋白胞外核心区(E2c)并利用E2c蛋白检测HCV患者血清中特异性抗E2蛋白抗体水平。方法以HCV1b型(河北株)基因序列为基础,设计HCV1b E2c基因序列并通过基因拼接的方法合成;将含有组织型纤溶酶原激活物(t PA)信号肽的E2c扩增产物克隆入p CI-neo真核表达载体,获得p CI-tpa-1b E2c载体;将p CI-tpa-1b E2c真核表达载体转染HEK293T细胞,收集细胞上清后进行浓缩及纯化,Western blot法鉴定蛋白特异性;以获得E2c蛋白为抗原,建立基于雪花莲凝集素(GNA)的改良ELISA检测HCV患者血清特异性抗E2c抗体水平。结果成功利用真核表达系统表达获得E2c蛋白,建立了GNA改良ELISA检测HCV患者血清中抗E2蛋白抗体的方法。结论成功在HEK293T细胞表达HCV-1b E2c蛋白并进行了临床应用。Objective To express core region of HCV1b（ Hebei strain） E2 protein（ E2c） by eukaryotic system,and establish the detection method of specific anti-HCV E2 antibody in the sera from hepatitis C patients. Methods Based on the literature,the E2 c gene was modified from the HCV1 b gene and synthesized via overlapping PCR. Thereafter,the E2 c gene including tissue-type plasminogen activator（ t PA） signal peptide was cloned into the p CI-neo eukaryotic expression vector,and the product was named p CI-tpa-1b E2 c. After HEK293 T cells were transfected with p CI-tpa-1b E2 c,the supernatant was collected,condensed and purified. Its specificity was identified by Western blotting. Galanthus nivalis agglutinin（ GNA）-based ELISA was used to detect the antibody against HCVE2 in the sera from hepatitis C patients. Results Modified HCV E2 c protein was successfully expressed in HEK293 T cells and the GNA-based ELISA was developed for detecting the antibody against HCV E2 in the sera from hepatitis C patients. Conclusion HCV-1b E2 c protein can be effectively expressed in HEK293 T cells and applied clinically.

关 键 词：丙型肝炎病毒(HCV) E2蛋白胞外核心区(E2c)蛋白 真核表达 基于雪花莲凝集素的改良ELISA 

分 类 号：R512.63[医药卫生—内科学] R392-33[医药卫生—临床医学]