无偿献血者隐匿性乙肝感染病毒变异检测分析

Analysis of virus mutation in blood donors with occult hepatitis B infection

目的通过对隐匿性乙肝感染者S区基因变异位点的分析及相关蛋白空间结构的预测,探讨无偿献血者隐匿性乙肝感染的分子生物学机制。方法对80例HBs Ag-/HBV DNA+无偿献血者进行血清学补充试验,并对追踪确认的隐匿性乙肝感染者,应用巢氏PCR技术对S区基因进行扩增和序列测定,并将测得基因片段与NCBI Gen Bank数据库做序列比对分析,明确突变位点,同时进行蛋白模型构建,进行空间结构的分析。结果 HBs Ag-/HBV DNA+无偿献血者血清学检测模式多样,以抗-HBc阳性为主,追踪试验确认4例隐匿性乙肝感染者,有3例巢氏PCR扩增出S区基因序列,2例发生突变,蛋白预测提示氨基酸改变可能影响抗原决定簇空间构象,造成抗体结合位点改变或识别能力减弱。结论隐匿性乙肝感染者S区容易发生变异,尤其是a抗原决定簇氨基酸的改变导致蛋白质空间构象的变化,可能是造成隐匿性乙肝发生的原因之一。

Objective To discuss the molecular biology mechanism of occult hepatitis B infection in unpaid donors by the analysis of gene mutation at area S in patients with occult hepatitis B infection and by the prediction of related protein space structure.Methods 80 samples of HBs Ag-/HBV DNA ＋ were conducted for supplement test; and blood donors with negative results were followed up,an Nested PCR was used for amplification and sequencing of genes in area S. Then sequence alignment analysis was carried out for the detected gene fragments with NCBI Gene Bank to clarify the definite mutation site,while construction of protein models and prediction of protein were carried out to analyze the space structure. Results Various serological modes was used in the detection of unpaid donors with HBs Ag-/HBV DNA ＋,which were given priority to positive anti-HBC. 4 cases with occult hepatitis B infection were confirmed by follow-up test,where area S gene sequences were amplified with nested PCR in 3 cases. Mutation analysis found mutation of amino acid was detected in area S of 2 cases. Protein prediction showed that amino acid mutations affected antigenic determinant space conformation,and thus leading to changes of antibody-combining site or reduction of identification ability. Conclusion Mutation in area S often occurs in patients with occult hepatitis B,especially changes of protein space structure due to changes of antigenic determinant amino acid,which might be one of the main causes of occult hepatitis B.