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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 Eukaryotic cells express the extracellular core (E2c) of E2 protein of Hepatitis C virus (HCV) and use E2c protein to detect the serum level of specific anti-E2 protein in HCV patients. Methods The HCV1b E2c gene sequence was designed based on the HCV1b (Hebei strain) gene sequence and was synthesized by gene splicing. The E2c amplification product containing the tissue plasminogen activator (t PA) signal peptide was cloned into p CI-neo eukaryotic expression vector to obtain pCI-tpa-1b E2c vector; pCI-tpa-1b E2c eukaryotic expression vector transfected HEK293T cells, the cell supernatant was collected after concentration and purification, Western blot identification of protein Specificity. To obtain E2c protein as antigen, a modified ELISA based on Snowdrop Lectin (GNA) was established to detect serum specific anti-E2c antibody in HCV patients. Results E2c protein was successfully expressed in eukaryotic expression system and the method of GNA modified ELISA was established to detect anti-E2 protein in serum of HCV patients. Conclusion The HCV E2b protein was successfully expressed in HEK293T cells and was used clinically.