【摘要】 构建了含一拷贝和两拷贝串连的PreS1(2 1～ 47aa)DNA片段与GST的融合表达质粒 pGEXSI与pGEXSII。经IPTG诱导 ,这两种质粒在大肠杆菌TG1中获得了高效表达。SDS PAGE结果表明 ,GST PreS1(2 1～ 47aa)和GST 2×PreS1(2 1～ 47aa)蛋白的表达量约分别占菌体总蛋白量的 30 %和 15 %。采用谷胱甘肽 Sepharose 4B凝胶 ,经一步亲和层析 ,可分别从 1L培养液的菌体裂解液中 ,纯化得到 5 0mg和 2 0mg纯度为 90 %以上的表达产物。直接ELISA的结果表明 ,含两个PreS1(2 1～ 47aa)片段的融合蛋白GST 2×PreS1(2 1～ 47aa)与抗PreS1(2 1～ 47aa)抗体的反应性比GST PreS1(2 1～ 47aa)和PreS1(2 1～ 47aa)合成肽强。以GST 2×PreS1(2 1～47aa)为包被抗原 ,通过生物素化A蛋白和HRP标记的链霉亲和素系统 ,建立了血清抗PreS1(2 1～ 47aa)抗体检测的间接ELISA。该法对近百名乙肝患者血清标本检测结果显示 ,急性乙肝病人中有近半数可检测到抗PreS1抗体 ,而对于慢性乙肝病人其阳性率则很低 ,统计分析表明急性和慢性乙肝病人抗PreS1抗体水平有显著性差异 (P<0 .0 1)。临床分析结果提示 ,抗PreS1抗体的出现与病程的进展及康复有一定的相关性 ,因而研究的结果有潜在开发应用的前景更多还原

【Abstract】 Expression plasmids pGEXSI and pGEXSII containing one copy and two orderly joined copies of PreS1(21—47 aa) DNA fragment, respectively, were constructed. GST PreS1(21—47 aa) and GST 2×PreS1(21—47 aa) fusion proteins were highly expressed in E.Coli TG1, induced by IPTG. The expression level of GST PreS1(21—47 aa) was about 30% of total soluble proteins in the lysate of expression bacteria, and GST 2×PreS1(21—47 aa) was about 15% of total soluble proteins, as estimated by SDS PAGE. 50 mg GST PreS1(21—47 aa) or 20 mg GST 2×PreS1(21—47 aa) with purity over 90% was obtained, respectively, from 1 L culture by using affinity chromatography of glutathione Sepharose 4B. Direct ELISA results showed that antigenicity of GST 2×PreS1(21—47 aa) was better than GST PreS1(21—47 aa) and synthetic peptide. Using GST 2×PreS1(21—47 aa) as coated antigen, a sensitive indirect ELISA for detection of anti PreS1(21—47 aa) antibody, based on protein A biotin and streptavidin HRP, was established. The results from 99 sera samples of hepatitis B patients showed that anti PreS1(21—47 aa) antibody was detected in nearly half of acute hepatitis B patients during recovery, but it was detected only in a few chronic hepatitis patients. Clinical follow up study suggested that appearance of anti PreS1(21—47 aa) was related to the course of the disease and recovery of patients. Detection system established in the study is promising for clinical application.更多还原