乙型肝炎病毒聚合酶片段的表达及其在血清学检测中的应用

Expression of DNA polymerase fragments of hepatitis B virus and its application in serological assay

目的建立乙型肝炎患者血清HBV病毒聚合酶抗体(抗-HBp)的ELISA检测方法。方法构建HBV反转录酶/DNA聚合酶(HBV-Pol)基因原核表达载体pQE30-Pol,表达并纯化目的蛋白,用间接ELISA法检测274例乙型肝炎患者及50例正常人血清中的抗-HBp抗体,并与HBV-DNA实时荧光定量检测结果比较。结果成功将HBV-Pol基因插入原核表达载体pQE30,并在大肠埃希菌M15中获得了高效表达,表达产物以包涵体形式存在。以纯化的重组蛋白建立间接ELISA检测方法,在检测的274例乙型肝炎患者中,HBsAg+/HBeAg+/抗HBc+,HBsAg+/抗HBe+/抗HBc+,HBsAg+/抗HBc+患者血清中的抗-HBp抗体阳性率分别为97.8%、57.9%和13.2%,平均阳性率58.8%,而正常人血清抗-HBp均为阴性。运用此法测得的血清抗-HBp阳性率和实时定量PCR检测结果无统计学差异(P=0.359)。结论成功表达了HBV-Pol片段并且建立了血清抗-HBp抗体的检测方法,为进一步研究乙型肝炎患者血清HBV-Pol抗体的血清学意义奠定了基础。

Objective To develop an indirect enzyme linked immunosorbent assay （ELISA） to detect anti-HBV-Pol antibody in serum of hepatitis B patients.MethodsThe coding sequence of HBV-Pol fragment was excised with BamH Ⅰ/Sal Ⅰ from a plasmid pUVN4 containing full length HBV adw2 subtype genome and sub-cloned into pQE30 to construct an prokaryotic expression plasmid pQE30-Pol.The target protein was expressed and purified.The purified recombinant antigen was coated on the microplate wells for the detection of anti-HBp antibody in serum by indirect ELISA.The ELISA results were analyzed and compared with those received by HBV real-time quantitative fluorescent detection.ResultsHBV-Pol gene was successfully cloned into prokaryotic expression plasmid pQE30,and HBV-Pol was highly expressed in E.coli M15 as an inclusion body.The indirect ELISA assay revealed that the positive rate of anti-HBp in HBsAg＋/HBeAg＋/anti-HBc＋,HBsAg＋/anti-HBe＋/anti-HBc＋ and HBsAg＋/anti-HBc＋ patients was 97.8%,57.9% and 13.2%,respectively,with a mean positive rate of 58.8% in 274 hepatitis B patients.There was no significant difference between the two methods as mentioned above （P=0.359）.ConclusionsRecombinant HBV-Pol fragment is produced and an indirect ELISA for anti-HBp detection has been developed.This indirect ELISA assay may lay a foundation for the study of clinical implications of serum anti-HBp.