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目的研究不同民族慢性乙型肝炎(CHB)患者HBV前S2/S(preS2/S)和C(core,C)基因在大肠杆菌Escherichia coli(E.coli)中表达的稳定性和水平,并对其抗原性进行鉴定,为创制新型疫苗提供抗原材料。方法从中国云南少数民族地区50例慢性乙型肝炎患者血清中提取HBV基因组DNA,将聚合酶链反应(PCR)扩增-克隆-测序的HBV preS2/S和C基因插入到表达载体pλPR上,构建重组pλPR-S2S和pλPR-C表达质粒,并在大肠杆菌TOP10中表达。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)测定表达产物非融合蛋白,并对其抗原性用Western印迹和ELISA方法进行检测。结果SDS-PAGE检测到全部慢性乙型肝炎患者pλPR-S2S和pλPR-C重组质粒在大肠杆菌TOP10中存在稳定性和高水平的表达,表达的非融合蛋白相对分子质量分别为31000和21000,非融合蛋白浓度约占16%,纯度达96%。Western印迹和ELISA分析,非融合蛋白能分别与HBVpreS2/S抗原单抗、C抗原单抗及慢性乙型肝炎患者血清发生反应,而与对照的甲型肝炎(HAV)患者血清、丙型肝炎(HCV)患者血清及正常血清之间无交叉反应。结论中国云南少数民族地区慢性乙型肝炎患者HBV preS2/S和C基因在大肠杆菌TOP10中有稳定性和高水平的表达,表达的非融合蛋白具有抗原性。这些发现为创制新型疫苗具有潜在应用价值。
Objective To study the stability and the level of preS2 / S and C (core, C) genes expressed in Escherichia coli (E.coli) of different ethnic groups with chronic hepatitis B Its antigenicity was identified, providing antigens for the creation of new vaccines. Methods HBV genomic DNA was extracted from sera of 50 patients with chronic hepatitis B in ethnic minority areas of Yunnan, China. The PCR products of preS2 / S and C were amplified by PCR and cloned into the expression vector pλPR. Recombinant pλPR-S2S and pλPR-C expression plasmids were constructed and expressed in E. coli TOP10. The expression product non-fusion protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and its antigenicity was detected by Western blotting and ELISA. Results The results of SDS-PAGE showed that the recombinant plasmids pλPR-S2S and pλPR-C of all patients with chronic hepatitis B had stable and high expression in E.coli TOP10. The relative molecular mass of the non-fusion proteins were 31000 and 21000, respectively Fusion protein concentration of about 16%, purity of 96%. Western blotting and ELISA analysis showed that non-fusion proteins reacted with serum of HBVpreS2 / S monoclonal antibody, C antigen monoclonal antibody and chronic hepatitis B patients, respectively, but not with serum of control hepatitis A (HAV) HCV) serum and normal serum no cross-reaction. Conclusions The HBV preS2 / S and C genes in patients with chronic hepatitis B in Yunnan ethnic minority areas in China are stable and highly expressed in E. coli TOP10. The expressed non-fusion protein has antigenicity. These findings have potential applications for the creation of new vaccines.