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目的建立检测患者血清中 HBV 的 x 抗原(HBxAg)及其抗体(anti-HBx)的方法,阐明其在慢性肝炎、肝硬化和肝癌患者血清中的临床意义。方法将 HBV 的 x 基因全长克隆至原核表达载体 pET30a(+)中,构建 pET30a-X 原核表达载体,转化入大肠杆菌 BL21(DE3)后,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达 His-HBx 融合蛋白,纯化后将重组蛋白免疫家兔,制备兔抗 HBx 多克隆抗体。应用纯化后的兔抗 HBx 多克隆抗体和纯化后的重组蛋白,建立检测血清中 HBxAg 和 anti-HBx 的方法。应用建立的 ELISA 法,分别对慢性乙型肝炎、肝硬化和肝癌患者组及正常对照组血清中 HBxAg 和 anti-HBx 进行检测。结果 HBxAg/anti-HBx 在慢性乙型肝炎、肝硬化和肝癌患者中的阳性率分别为8.7%/10.4%,17.9%/40.6%,9.8%/34.4%。统计学分析显示,与慢性乙型肝炎相比,肝硬化或肝癌患者血清中 anti-HBx 的阳性率较高(P<0.01)。结合临床检测的其他 HBV 血清学指标,发现在 anti-HBs 阳性病例中,anti-HBx 阳性率为5.2%(1/19);而在 HBsAg、HBeAg 和 anti-HBc 3项同时为阳性的病例中,anti-HBx 的阳性率为28.9%(39/135),二者相比,差异具有统计学意义(P<0.05)。213名正常对照血清中 HBxAg 和 anti-HBx 均为阴性。结论血清中 anti-HBx 并非一个保护性抗体,标志 HBV 的感染和复制,是从慢性乙型肝炎向肝硬化和(或)肝癌发展的血清标志之一。
Objective To establish a method for the detection of HBxAg and anti-HBx in patients with sera and to clarify its clinical significance in the serum of patients with chronic hepatitis, cirrhosis and liver cancer. Methods The full-length x gene of HBV was cloned into the prokaryotic expression vector pET30a (+) to construct the prokaryotic expression vector pET30a-X. The recombinant plasmid was transformed into E. coli BL21 (DE3) Gly-glycoside (IPTG) induced the expression of His-HBx fusion protein and purified the recombinant protein to immunize rabbit to prepare rabbit anti-HBx polyclonal antibody. The purified rabbit anti-HBx polyclonal antibody and the purified recombinant protein were used to establish a method for the detection of HBxAg and anti-HBx in serum. The HBsAg and anti-HBx levels in sera of patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma and normal controls were detected by ELISA. Results The positive rates of HBxAg / anti-HBx in patients with chronic hepatitis B, liver cirrhosis and liver cancer were 8.7% / 10.4%, 17.9% / 40.6% and 9.8% / 34.4% respectively. Statistical analysis showed that compared with chronic hepatitis B, cirrhosis or liver cancer serum anti-HBx positive rate was higher (P <0.01). In combination with other HBV serological tests, the positive rate of anti-HBx was 5.2% (1/19) in anti-HBs-positive cases and in the case of both positive HBsAg, HBeAg and anti-HBc , the positive rate of anti-HBx was 28.9% (39/135), the difference was statistically significant (P <0.05). 213 normal control serum HBxAg and anti-HBx were negative. Conclusion Serum anti-HBx is not a protective antibody, marking the infection and replication of HBV. It is one of the serum markers for the development of cirrhosis and / or liver cancer from chronic hepatitis B.