摘要
目的本研究旨在发现和研究具有潜在生物学或临床意义的乙型肝炎小表面蛋白(SHBs)新突变。方法SHBs氨基酸(AA)序列来自本课题组数据库227例患者,患者为未经抗病毒治疗、e抗原阳性的慢性乙型肝炎(慢乙肝)患者,均感染C2基因亚型乙肝病毒(hepatitis B virus,HBV)。通过SHBs的AA突变与血清乙型肝炎表面抗原(HBsAg)水平的关联分析,发现可能影响血清HBsAg水平的新突变。用重叠PCR法构建突变质粒,转染HepG2肝癌细胞系,用ELISA和Western Blot法检测HBsAg,荧光定量PCR检测胞内的HBV总RNA水平和细胞内外HBV DNA水平。结果SHBs序列突变检出率为78.4%(178/227),仅在高水平HBsAg组中检出的sC76Y(5/113,4.4%)和sI218L(3/113,2.7%)为新突变。与野生型相比,sC76Y突变株可使胞内HBsAg、胞内HBV总RNA和胞外HBV DNA明显升高(P<0.05),胞外HBsAg和胞内HBV DNA显著降低(P<0.05)。sI218L突变株可使胞内HBV总RNA显著增加(P<0.05),并使胞外HBsAg水平显著改变。结论新发现的sC76Y和sI218L突变可能通过不同机制影响HBV复制。前者倾向于使HBsAg滞留胞内,但可能促进病毒体分泌;后者则与胞内较高水平HBV RNA有关,还可能影响HBsAg的抗原性。
Objective To explore and study the novel amino acid(AA)mutations in small hepatitis B surface protein(SHBs)sequences.Methods The AA sequences of SHBs in our database were obtained from 227 patients of HBeAg positive chronic hepatitis B without treatment.They were all infected with genotype C2hepatitis B virus(HBV).The association analysis on AA mutation and serum hepatitis B surface antigen(HBsAg)level was applied to reveal the novel SHBs mutations of interest.The mutant plasmids were constructed by overlap PCR.The HepG2cell line was used for in vitro study.HBsAg,HBV total RNA and HBV DNA was measured by ELISA/Western Blot,and real time quantitative PCR,respectively.Results Of the 227patients,SHBs sequences was found to harbor AA mutations in 178patients(78.4%)and most of the mutations affecting serum HBsAg levels have been reported in previous studies;however,sC76Y(5/113,4.4%)and sI218L(3/113,2.7%)mutations were only detected in the HBsAg high level group and thus worthwhile for in vitro study.Comparing to the wild type,the sC76Y mutant displayed significantly higher intracellular HBV HBsAg and HBV total RNA,extracellular HBV DNA(P<0.05);but significantly lower extracellular HBsAg and intracellular HBV DNA(P<0.05).The sI218Lmutant only showed a significantly higher HBV total RNA(P<0.05).The intracellular and extracellular HBsAg levels of sI218Lwere also found to be different from those of the wild type.Conclusions The novel mutations of sC76Yand sI218Lcould affect HBV replication through potentially different mechanisms.The former is prone to maintain HBsAg within the cells and potentially enhance the virion secretion.The latter seems to be associated with a higher level of intracellular HBV RNA and a changed antigenicity of HBsAg.
作者
王璐薇
苏明泽
欧国敏
和凌媛
李垚
庄辉
向宽辉
李彤
WANG Lu-wei;SU Ming-ze;OU Guo-min;HE Ling-yuan;LI Yao;ZHUANG Hui;XIANG Kuan-hui;LI Tong(Department of Microbiology and Infectious Disease Center,School of Basic Medical Sciences,Peking University Health Science Center,Beijing 100191,China)
出处
《中国病毒病杂志》
CAS
2020年第3期175-183,共9页
Chinese Journal of Viral Diseases
基金
国家“十三五”艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2017ZX10202202-004-004)
国家自然科学基金面上项目(81772174)。
