摘要
目的自从建立起甲、乙、丙、丁、戊5种肝炎病毒的病原学诊断之后,仍有少部分肝炎患者的病因得不到明确,因此不少学者试图探索是否还有新型肝炎病毒的存在,并进行了大量的流行病学和实验诊断的研究,认为的确存在可经肠道外传播并引起人类肝炎的致病因子.1995年 Simons et al 成功地克隆出2株黄病毒 RNA 序列,称之为 GBV-A,GBV-B.继而从一名西非患者血清中克隆出另一黄病毒 RNA 序列,称之为 GBV-C.1996年初美国另一研究小组从一名慢性输血后肝炎患者血清中克隆出一株黄病毒 RNA 序列,称之为 HGV.进一步的研究后认为 GBV-C,HGV 为同一病毒的不同分离株.动物实验发现,GBV-B 可单独引起动物发病,而 GBV-A 单独感染时未能引起发病.本文以此为依据,对 GBV-B 感染者的临床与免疫病理加以研究,试图探讨 GBV-B 对人体肝脏的致病性及其在人体内的分布状态.方法根据 GBV-B NS5区的核苷酸和氨基酸序列,通过计算机软件对其抗原位点进行了预测,并分析其亲水性、键流动性、电荷状态及抗原表位分布等特性,合成一条30个氨基酸多肽.探讨合成肽最佳包被量之后,成功地建立了间接 ELISA 法,用来检测抗-GBV-B.应用此方法检测各型肝炎及其他高危人群血清标本1286份.同时应用 RT-PCR 检测血清中 GBV-B RNA及免疫组化检测肝炎患者肝组织相关病毒抗原.结果经鉴定合成肽纯度在95%以上,氨基酸分析的实际值与理论值基本一致.将合成肽与牛血清清蛋白(BSA)偶联免疫白兔获得高效价抗-GBV-B NS5区的多克隆抗体(PcAb).批内、批间变异系数分别为6.06%和7.65%(均<10%);中和抑制试验结果证明我们建立的 ELISA 法具有较好的特异性.血清抗-GBV-B 检测结果表明各型肝炎患者血清抗-GBV-B 阳性率为10.56%;其中非甲～非戊型肝炎患者血清抗体阳性率为8.57%;肝炎高危人群如献血员、血液透析者及静脉药瘾者血清抗-GBV-B 阳性率分别为2.90%,8.62%及13.44%.随机抽取抗-GBV-B 阳性和抗-GBV-B 阴性血清标本各32例进行RT-PCR 检测 GBV-B RNA,结果64例均为阴性.应用抗-GBV-B 多克隆抗体为试剂,对42例肝炎患者肝组织进行免疫组化研究,均未检测出 GBV-B 相关抗原.结论提示 GBV-B 可能不是人类肝炎病毒,对人体肝脏的致病性轻微,其抗体在不同人群中出现可能是一种短暂的感染,其意义有待进一步研究.
AIM To study the clinical features and pathological changes of individuals infected with GBV-B in order to evaluate the pathogenicity of GBV-B in human liver and its distribution state in human tissues based on the discovery described previously. METHODS According to the nucleotide and amino acid sequence of GBV-B NS5 region,a 30 amino acids polypeptide was synthesized following the prediction for the antigen epitopes by computer and analysis of its hydrophilicity,bond fluidity,charge condition and epitopes distribution.An indirect enzyme-linked immunoabsorbent assay (ELISA) detecting antibodies against GBV-B was developed after having tested the optimal coating dose.Using the developed ELISA,the seroprevalence of antibodies against GBV-B in 1286 sera from patients with various types of hepatitis and populations at high risk was determined.GBV-B RNA and liver tissue of hepatitis patients were detected with RT- PCR and immunohistochemistry respectively. RESULTS The purity of the synthetic peptide was up to 95%.The practical and theoretical value for amino acid analysis was identical approximately.A high titer polyclonal antibody (PcAb) to GBV-B NS5 region epitopes was obtained from rabbits immunized with the synthetic peptide coupled with BSA.Intrabatch and interbatch coefficients of variation were 6.06% and 7.65% respectively (all below 10%).Neutralizing inhibition test indicated that the specificity of our ELISA was satisfactory.The seroprevalence in various types of hepatitis was 10.56% with respect to GBV-B,whereas a seroprevalence of 8.57% was observed in non-A-E hepatitis patients.The seropositivity in high-risk poulations such as blood donors,patients undergoing blood dialysis and intravenous drug abusers was 2.90%, 8.62%,and 13.44% respectively.Thirty-two anti-GBV-B positive and 32 anti-GBV-B negative sera were randomly selected and tested for the presence of GBV-B RNA using RT-PCR,but none was detected in all 64 serum specimens.In addition,GBV-B associated antigen was not detectable in liver tissues of 42 hepatitis patients by immunohistoch- emistry using anti-GBV-B polycolonal antibody. CONCLUSION GBV-B may not be a human hepatitis virus, causing minimal pathological change of liver.The presence of anti-GBV-B at different populations might denote a temporary infection,and its implications remain to be further studied.
出处
《世界华人消化杂志》
CAS
2000年第7期775-781,共7页
World Chinese Journal of Digestology
关键词
GB病毒B型
合成肽
ELISA
免疫组化
GB virus B(GBV-B)
infection
synthetic peptide
ELISA
RT-PCR
immunohistochemistry
