抗丙型肝炎病毒E2蛋白人单链Fv抗体的制备及其免疫组织化学鉴定
Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry.
作者信息
Zhong Yan-Wei, Cheng Jun, Wang Gang, Shi Shuang-Shuang, Li Li, Zhang Ling-Xia, Chen Ju-Mei
机构信息
Gene Therapy Research Center, Institute of Infectious Diseases, 302 Hospital of PLA, 26 Fengtai Road, Beijing 100039, China.
出版信息
World J Gastroenterol. 2002 Oct;8(5):863-7. doi: 10.3748/wjg.v8.i5.863.
AIM
To screen human single chain Fv antibody (scFv) against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.
METHODS
The phage antibody library was panned by HCV E2 antigen, which was coated in microtiter plate. After five rounds of biopanning,56 phage clones were identified specific to HCV E2 antigen. The selected scFv clones were digested by SfiI/NotI and DNA was sequenced. Then it was subcloned into the vector pCANTAB5E for expression as E-tagged soluble scFv. The liver tissue sections from normal person and patients with chronic hepatitis B and chronic hepatitis C were immunostained with HCV E2 scFv antibody.
RESULTS
The data of scFv-E2 DNA digestion and DNA sequencing showed that the scFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis C E2 antigen has a specific binding character with hepatitis virus E2 antigen and paraffin-embedded tissue, but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.
CONCLUSION
We have successfully screened and identified HCV E2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitis C.
目的
筛选抗丙型肝炎病毒E2抗原的人单链Fv抗体（scFv），并鉴定其在免疫组织化学中的应用。
方法
用包被于微量滴定板的丙型肝炎病毒E2抗原淘选噬菌体抗体库。经过五轮生物淘选，鉴定出56个对丙型肝炎病毒E2抗原有特异性的噬菌体克隆。将所选的scFv克隆用SfiI/NotI酶切并进行DNA测序。然后将其亚克隆到载体pCANTAB5E中以表达带E标签的可溶性scFv。用丙型肝炎病毒E2 scFv抗体对正常人以及慢性乙型肝炎和慢性丙型肝炎患者的肝组织切片进行免疫染色。
结果
scFv-E2 DNA酶切和DNA测序数据表明scFv基因由750 bp组成。酶联免疫吸附测定（ELISA）和免疫组织化学表明，抗丙型肝炎病毒E2抗原的人单链Fv抗体与丙型肝炎病毒E2抗原和石蜡包埋组织具有特异性结合特性，但与健康人或慢性乙型肝炎患者的肝组织无反应。
结论
我们成功筛选并鉴定出丙型肝炎病毒E2 scFv，该scFv可用于慢性丙型肝炎患者肝组织切片的免疫染色。
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