Bo Hu, Minjian Liang, Guoqiang Hong, Zhaoxia Li, Zhenyu Zhu, Lin Li
Department of Laboratory Medicine, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province,510630, China.
J Biochem Mol Biol. 2005 Nov 30;38(6):683-9. doi: 10.5483/bmbrep.2005.38.6.683.
Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying a hexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5 % of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100 % with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.
乙型肝炎表面抗原抗体（HBsAb）是乙肝病毒（HBV）感染的重要血清学标志物。传统上，从HBV携带者血浆中获取的乙型肝炎表面抗原（HBsAg）用作检测HBsAb的诊断抗原。这种血液来源的抗原存在一些缺点，包括成本高、制备繁琐、有感染风险等。为了探索适合诊断用途的重组HBsAg，将HBV S基因在毕赤酵母中表达，并将产物用于检测HBsAb。将乙肝病毒S基因插入酵母载体，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹、电子显微镜和酶联免疫吸附测定（ELISA）对表达产物进行分析。通过夹心ELISA将合成的S蛋白制剂用于检测HBsAb。编码带有C末端六组氨酸标签的226个氨基酸的HBsAg的S基因在毕赤酵母中成功表达。该菌株中带有His标签的S蛋白表达量约占总细胞蛋白的14.5%。免疫印迹显示重组HBsAg可被单克隆HBsAb识别，宿主的所有蛋白与正常血清之间无交叉反应。HBsAb检测表明，按照国家临床检验中心的HBsAb标准，灵敏度达到10 mIu（微国际单位）/ml，特异性为100%。使用重组S蛋白和市售HBsAb ELISA试剂盒（由血液来源的HBsAg生产）对总共293份随机血清进行检测，检测了35份HBsAb阳性血清和258份HBsAb阴性血清。两种不同试剂得到相同结果，两种试剂之间的S/CO值无显著差异。具有良好免疫反应性和特异性的重组HBV S蛋白在毕赤酵母中成功表达。由毕赤酵母来源的S蛋白制备的用于检测HBsAb的试剂对HBsAb标准检测显示出高灵敏度和特异性。在随机样本中，重组S蛋白生产的试剂与血液来源的HBsAg生产的市售试剂盒之间具有良好的相关性。
