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目的 构建乙型肝炎病毒 (HBV)变异s基因真核表达载体 ,研究变异所致乙肝表面抗原 (HBsAg)免疫学性状的改变。方法 利用DraⅢ和XhoⅠ双酶切含有突变位点的HBVDNA 1.2拷贝的质粒P3.8Ⅱ ,获得 90 4bp带有突变点的HBV S基因片段。将其置换含有HBVDNA(adr亚型 )S和S2片段的真核表达载体 pCMV S2 .S的相应片段 ,从而构建s基因nt5 87G→A突变的真核表达载体 pCMV S2 .S +14 5R ;并转染人肝癌细胞系HepG2 ,以获得分泌变异型HBsAg的细胞系。利用EIA及细胞免疫染色法 ,探讨变异抗原与抗 HBs结合力的变化。结果 构建了HBsAg的第 14 5位甘氨酸→精氨酸突变体的真核表达载体。将其转染哺乳动物细胞后第 3天 ,培养上清中用EIA检测HBsAg呈阳性 ,A值随时间延长而上升 ,但变异型的A值明显低于野生型。变异型与野生型HBsAg细胞免疫化学检测均有部分细胞呈阳性 ,但差别不显著。结论 HBsAg第 14 5位甘氨酸→精氨酸的突变体的真核表达载体表达产物具有良好的抗原性 ,能够与抗 HBs结合 ,但与野生型相比结合力明显降低
Objective To construct eukaryotic expression vector of hepatitis B virus (HBV) s gene and study the change of immunological characters of hepatitis B surface antigen (HBsAg) induced by mutation. Methods The plasmid pSBPII of HBV DNA containing 1.2 bp of HBVDNA was digested with Dra Ⅲ and Xho Ⅰ to obtain 90 bp of HBV S gene fragment with mutation point. The eukaryotic expression vector pCMV S2 .S +14 5R with the nt5 87G → A mutation of s gene was constructed by replacing the corresponding fragment of the eukaryotic expression vector pCMV S2 .S containing the S and S2 fragments of HBVDNA (adr subtype) with Human hepatoma cell line HepG2 was transfected to obtain a cell line secreting mutant HBsAg. EIA and cell immunostaining were used to investigate the binding capacity of antigens to anti-HBs. Results The eukaryotic expression vector of glycine → arginine at position 145 of HBsAg was constructed. On the third day after transfection into mammalian cells, HBsAg was detected by EIA in culture supernatant. The value of A increased with time, but the value of variant A was significantly lower than that of wild type. Immunochemical detection of variant and wild-type HBsAg cells showed some cells were positive, but the difference was not significant. CONCLUSION: The eukaryotic expression vector of glycine to arginine at position 14 5 of HBsAg has good antigenicity and can bind to anti-HBs, but its binding ability is obviously lower than that of wild-type