摘要
目的探讨丙型肝炎病毒（HCV）F蛋白的抗原性及患者血清F抗体的流行率。方法利用11条引物延伸获得HCVf基因，以此为模板经聚合酶链反应扩增得到截短型f65基因片段，定向克隆至pET32a（＋），重组子转化大肠杆菌Plyss菌株，异丙基-β-半乳糖苷诱导后用镍离子树脂纯化HCVF65蛋白。以F65蛋白为抗原，酶联免疫吸附法检测HCV感染者血清中F抗体。应用F65蛋白免疫新西兰大白兔制备多克隆F抗体，再用葡萄球菌A蛋白树脂纯化兔血清中F抗体。结果成功构建了pET32a（＋）f65重组子，表达并纯化了分子量为3．2×10^4的HCVF65蛋白。30份HCV患者血清F抗体的A450为0．125±0．061，F抗体阳性率为63．3％。获得了兔源性F抗体，该抗体与HCVF65蛋白发生特异性结合反应，滴度为1：30000。结论HCVF65蛋白具有抗原性，可用来检测血清中F抗体。HCV患者血清中存在F抗体。兔源性F抗体可用来检测HCVF蛋白。
Objective To examine the antigenicity of hepatitis C virus （HCV） F protein and investigate serum prevalence of anti-F in HCV-infected patients. Methods Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a（＋）, and transformed into E.coli strain Plyss （DE3）. This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti- F, and to immunize rabbits for making polyclonal anti-E The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose. Results After recombinant pET32a（＋）-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients （n = 30）. The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125 ± 0.061 and 63.3%, respectively. The rabbitderived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1 ： 301300. Conclusion Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-E Anti-F IgG does exist in the sera of the HCV infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2006年第12期890-893,共4页
Chinese Journal of Hepatology
基金
军队医药卫生"十一五"专项项目(06Z026)
军队医药卫生科研课题(04M017)
关键词
肝炎病毒
丙型
抗原
病毒
抗体
病毒
F蛋白
Hepatitis C virus
Antigens, viral
Antibodies, viral
F protein
