摘要
目的 通过构建HBV C基因型突变质粒研究HBsAg阴性HBV DNA阳性患者HBV S区突变对HBsAg水平的影响.方法 收集2022年8月至2023年4月首都医科大学附属北京佑安医院107例HBsAg-/HBV DNA+患者血液样本,对成功提取扩增的HBV DNA S区进行测序,通过构建HBV C基因型突变质粒对HBV S区突变位点进行细胞功能验证,探讨OBI可能发生的分子机制.结果 对成功提取扩增的68例患者进行测序,发现HBV S区存在大量突变,包括免疫逃逸突变(如sG145R、sK122R、sS114T、sT131P等)和跨膜结构域(transmembrane domain,TMD)突变(如sT5A、sG10D、sF20S等).通过构建HBV C基因型突变质粒,进行细胞转染和细胞免疫荧光实验发现sG145R突变会明显降低HBsAg的表达,但是sK122R、sI26N、sQ29N、sR169H、sS114T、sT131P这6个突变位点并未影响细胞内外HBsAg的表达.结论 通过测序发现HBsAg-/HBV DNA+患者HBV S区存在大量突变位点,通过构建sG145R、sK122R、sI26N、sQ29N、sS114T和ST131P等突变质粒发现sG145R突变会明显降低细胞内外HBsAg的表达,但是sK122R、sI26N、sQ29N、sR169H、sS114T、sT131P并未明显降低细胞内外HBsAg的表达.
Abstract
Objective To study the effect of HBV S region mutation on HBsAg content in HBsAg negative HBV DNA positive patients by constructing HBV C genotype mutant plasmid.Methods A total of 107 HBsAg-/HBV DNA+patients from Beijing Youan Hospital,Capital Medical University from August,2022 to April,2023 were collected for the study,and the successfully amplified HBV DNA S region was sequenced.The cell function of the mutation sites in the HBV S region was verified by constructing HBV C genotype mutation plasmid to explore the molecular mechanism of the possible occurrence of OBI.Results Sequencing results of 68 patients showed that there were a large number of mutations in the S region of HBV,including immune escape mutations (such as sG145R,sK122R,sS114T,sT131P,etc.)and transmembrane domain (TMD) mutations (such as sT5A,sG10D,sF20S,etc.).We found that the sG145R mutation significantly reduced the expression of HBsAg,while six mutation sites of sK122R,sI26N,sQ29N,sR169H,sS114T,and sT131P did not affect the expression of HBsAg intracellularly or extracellularly.Conclusion In this paper,a large number of mutation sites are identified existing in the HBV S region of HBsAg-/HBV DNA+ patients by sequencing technique,and the mutant plasmids such as sG145R,sK122R,sI26N,sQ29N,sS114T and ST131P show that sG145R mutations significantly reduce the expression of HBsAg intracellularly or extracellularly,while sK122R,sI26N,sQ29N,sR169H,sS114T,and sT131P do not significantly reduce the expression of HBsAg in both intracellular and extracellular manner.
基金项目
国家科技重大专项(2018ZX10305-410-006)
北京市优秀人才项目(2018000021469G287)
首都医科大学附属北京佑安医院中青年人才孵育项目(BJYAYY-CY2019-11)
首都卫生发展科学研究专项公共卫生项目(2021-1G-4302)
维普
万方数据