表面抗原插入突变乙型肝炎病毒免疫逃避株的生物功能学分析  

Functional analysis of hepatitis B virus immune escape mutants with insertion mutations in the surface antigen

作　　者：于德敏[1] 张东华[1] 姜节洪[1] 陈嘉[1] 邓琳[1] 张欣欣[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院感染科,上海200025

出　　处：《中华肝脏病杂志》2013年第7期510-513,共4页Chinese Journal of Hepatology

基　　金：基金项目：国家自然科学基金（81171616）l上海科委国际合作项目（11410701700）

摘　　要：目的我们在121例血清HBsAg／抗一HBs双阳性HBV感染者中发现2例HBsAg编码区114-115位氨基酸插入突变，本研究分析了其中1种插入突变（NTT）对病毒生物学特性包括抗原性和复制能力的影响。方法利用生物信息学分析技术对发生插入突变的HBsAg的抗原性改变进行模拟分析，同时构建带有该插入突变的复制型质粒，体外表型研究突变株的抗原性和复制能力变化。结果HBsAg114-115位氨基酸的插入突变对HBsAg的抗原性指数、亲水性、表面可能性和蛋白骨架柔韧区域均产生影响，导致抗HBsAg抗体对抗原亲和力降低，但病毒的复制能力无明显改变。结论HBsAg114-115位的NTT氨基酸插入突变可导致病毒的免疫逃避，同时维持了病毒的正常复制能力。[Abstract] Objective To evaluate the influence of insertion mutations occurring in the hydrophobic region, between amino acids 114 and 115, of the hepatitis B surface antigen （HBsAg） on viral antigenicity and replication. Methods Hepatitis B virus （HBV） DNA was obtained from patients with HBsAg-positive chronic hepatitis B （CHB） infection and subjected to sequence analysis and comparison to GenBank reference sequences for HBV genotype B （AB073826） and genotype C （AF286594）. Insertion mutations detected in the HBsAg region were used to make recombinant expression plasmids via site- directed mutagenesis. After transfecting the recombinant HBsAg into Huh7 cells, the mutants＇ effects on viral antigenicity and replication were evaluated by chemiluminescence microparticle immunoassay （CMIA） and Southern blot hybridization, respectively. The viral antigenieity of each mutant was predicted by bioinformatic analysis, using the Jameson-Wolf method to predict the antigenic index, the Hopp- Woods method to predict hydrophilicity, the Emini method to predict the probability of a region lying of the protein＇s surface, and the Karplus-Schulz method to predict the flexibility of the protein backbone. Results Two CHB patients harbored HBV with insertion mutations in HBsAg： one with two （NT） and one with three （NTT） inserted amino acids between 114 and 115. The NTT recombinant HBsAg mutant showed no impact on viral replication and reacted weakly with anti-HBs in CMIA （P = 0.02）. The antigen indices for the insertion of NTT were 1.00, -0.16, and 0.18, and insertion of the three amino acids affected the index values of five proximal amino acid sites （with an average increase of 0.13）. The hydrophilic indices for the insertion of NTT were 0.2, -0.4, and -0.4, with no significant effect on the proximal amino acids. The insertion of the three amino acids changed both the surface probability （range： -0.55 to 2.97;affecting eight proximal amino acids） and the flexibility （range： -0.

关 键 词：肝炎病毒 乙型 肝炎表面抗原 乙型 突变 

分 类 号：R512.62[医药卫生—内科学]