丙型肝炎病毒F蛋白抗原性及患者血清F抗体流行率的研究

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条引物延伸获处HCV f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HCV F65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降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HCV F65蛋白。30份HCV患者血清F抗体的A_(450)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HCV F65蛋白发生特异性结合反应,滴度为1：30000。结论HCV F65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HCV F蛋白。

Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients

Objective To examine the antigenicity of hepatitis C virus(HCV)F protein and investigate serum preva- lence 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 polyclonat anti-F.The rabbit polyclonal anti-F was purified by Staphylococcus anreus 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 A_(450)value and the positive rate of serum anti-F were 0.125+0.061 and 63.3%,respectively.The rabbit- derived polyclonal anti-F reacted specifically with HCV F65 protein,of which the titer was 1:30 000.Conclusion Our expressed HCV F65 protcin is of antigenicity,and can be used to determine serum anti-F.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.