CAJ | 학술논문

目的我们在121例血清HBsAg/抗-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 HBsAg 114-115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 HBsAg 114-115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.
목적 아문재121례혈청HBsAg/항-HBs쌍양성HBV감염자중발현2례HBsAg편마구114-115위안기산삽입돌변,본연구분석료기중1충삽입돌변(NTT)대병독생물학특성포괄항원성화복제능력적영향. 방법 이용생물신식학분석기술대발생삽입돌변적HBsAg적항원성개변진행모의분석,동시구건대유해삽입돌변적복제형질립,체외표형연구돌변주적항원성화복제능력변화. 결과 HBsAg 114-115위안기산적삽입돌변대HBsAg적항원성지수、친수성、표면가능성화단백골가유인구역균산생영향,도치항HBsAg항체대항원친화력강저,단병독적복제능력무명현개변. 결론 HBsAg 114-115위적NTT안기산삽입돌변가도치병독적면역도피,동시유지료병독적정상복제능력.
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 sitedirected 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 antigenicity of each mutant was predicted by bioinformatic analysis,using the Jameson-Wolf method to predict the antigenic index,the HoppWoods 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:-0.01 to 1.1; affecting five proximal amino acids).Conclusion The NTT three amino acids insertion in the HBsAg open reading frame,between 114 and 115 of the wild-type sequence,detected in a CHB patient may play a role in HBV immune escape without influencing the viral replicative capacity.