乙型肝炎病毒X蛋白定量试验与临床研究

Hepatitis B Virus X Protein Quantitative Assay and Aplication Research

HBVX蛋白(HBx)是由乙型肝炎病毒(HBV)基因组编码的调控蛋白,与由乙肝引起的肝癌发生具有密切的关系。血清HBx对乙型肝炎、肝癌的诊断及发病机理研究有较高的临床应用价值。在表达并纯化HBx、制备出HBx单克隆抗体与酶标抗体的基础上,研制了HBx定量检测试剂盒(增强化学发光法),对其灵敏度、特异性等指标进行分析,并将该试剂盒应用于临床研究。结果表明,原核表达并纯化的HBx纯度≥94%;试剂盒灵敏度达0.1ng/ml;线性范围达到0.5～600ng/ml;特异性高,与球蛋白、脂蛋白、血红蛋白、酸性糖蛋白、HBc、HBe、HBs、HBV preS2不发生交叉反应;批间CV≤6.5%;临床标本检验慢性乙型肝炎阳性率为55%、肝硬化阳性率为68%、肝癌阳性率70%,表明此试剂盒可应用于乙肝、肝硬化、肝癌的临床诊断及发病机理研究。

Hepatitis B virus X （HBx） was a regulatory protein and it was coden by hepatitis B virus （HBV） genome. Serum HBx quantitative detection may be helpful for HBV, hepatocirrhosis, and hepatoma diagnosis. A HBx chemiluminesscence quantitative assay kit was produced following the recombinant HBx antigen expressed in E. Coli and the HBx monoclonal antibodies selected from HBx antigen immunizing mice. The sensibility, specificity and other characteristics of the HBx quantitative assay kit were analyzed. The purity of the recombinant HBx protein was ≥ 94%. Two of six monoclonal antibodies were selected and used for bait and detect antibody. The sensibility of the HBx quantitative assay kit is 0.1 ng/ml, the linearity range of it is 0.5 600ng/ml. No eoss-reactions to GLB, lipoprotein, HGB, HBe, HBe, HBs or HBV preS2 were observed. The addition accuracy was between 98.7% - 105.7% , and the dilution accuracy was between 95.4% - 104.5%. CV （within batch） ≤2.9, CV （batch to batch） ≤6.5. After keep in 37℃ for 4 days, the technical datas were almost same as keeping in 4℃. The HBx quantitative assay kit was use to detecte the HBx in 75 chronic HBV, 20 acute HBV, 25 hepatocirrhosis, 30 hepatoma patients＇ serum and 180 healthy people＇ s serum. More than 55% chronic HBV patients, 68% hepatocirrhosis patients and more than 70% hepatoma patients can be detected. The higher grade of hepatocirrhosis patients classification it was, the higher concentration of the serum HBx was being. All the results show that the HBx chemiluminesscence assay kit we developed can not only be used for serum HBx quantitative detection, but also be used for the relationship between HBx and the hepatoma or the hepatocirrhosis study.