血清HBeAg阴性与HBeAg/IC及A1896变异的关系
西安医科大学第一临床医学院肝炎研究室 710061
项目负责人 张树林，710061，西安医科大学第一临床医学院肝炎研究室.
Correspondence to Shu-Lin Zhang, Hepatitis Lab, First Clinical College, Xi＇an Medical University,Xi＇an 710061, Shaanxi Province, China
收稿日期 1998-07-09
Relation ship between negative serum HBeAg-and formation of HBeAg-specific immune complex and HBV mutant A1896
Shu-Lin Zhang, Jiang Zhu, Min Liu and Ning-Li Chai
Hepatitis Lab, First Clinical College, Xi＇an Medical University, Xi＇an 710061, Shaanxi Province, China
Abstract
AIM To explore the relation between serum HBeAg-negative and HBeAg-specific immune complex (HBeAg/IC)， HBV mutant A1896, to evaluate the clinical significance of HBeAg/IC detetion.
METHODS The serum HBeAg/IC was deteted by ELISA using monolonal antibody against HBeAg. The serum HBV DNA was tested with nested polymerase chain reaction (n-PCR) using nested primers induced from S region of HBV genome. A1896 mutant was determined by 3＇-base specific PCR (3＇-BS-PCR). In this study, 117 patients with chronic HBV infection were investigated. Statistical analysis of data was conducted with X test.
RESULTS The detection rate of serum HBV DNA was greatly higher in sera with positive HBeAg/IC than that in sera with negative HBeAg/IC P<0.001 (91.3% vs 36.2%). Twenty nine patients with positive HBV DNA had no HBeAg in serum, only 5 of them (17.2%) were infected with A1896mutant, but 2 of the 5 patients also had wild type HBV (G1896) and HBeAg/IC in sera, 17 patients (58.7%) were infected with G1896 and had HBeAg/IC in serum. The prevalence of A1896 was much higher in sera with positive anti-HBe than that in sera with negative anti-HBe.
CONCLUSION HBeAg/IC is one of the markers indicating HBV active replication. Most patients with negative HBeAg and positive HBV DNA in serum are infected with wild type HBV (G1896)， the formation of HBeAg/IC results in undetection of HBeAg with “Sandwich” ELISA: Anti-HBe response may be one of the factors that promote the mutation of pre-C gene of HBV.
Subject headings hepatitis, viral; HBeAg; hepatitis B
Zhang SL, Zhu J, Liu M, Chai NL. Relation ship between negative serum HBeAgand formation of HbeAg-specific immune complex and HBV mutant A 1896. Huaren Xiaohua Zazhi,1998;6(11):960-962
摘要
目的 探讨血清HBeAg阴性(双抗体夹心法)与HBeAg/IC形成及HBV变异株A 1896的关系，评价HBeAg/IC检测的临床意义.
方法 单克隆抗HBe固相ELISA检测血清中HBeAg/IC；套式多聚酶链反应检测HBVDNA；3＇碱基特异多聚酶链反应判断A 1896变异；ELISA检测HBeAg、抗HBe，研究对象为117例慢性HBV感染者，20例健康对照统计处理采用卡方检验.
结果 HBeAg/IC阳性血清中HBV DNA检出率明显高于HBeAg/IC阴性血清，P<0.001(91.3% vs 36.2%)；29份HBeAg阴性、HBVDNA阳性血清中仅5例(17.2%)检出A 1896，而且其中2例与野毒株(G 1896)混合感染并伴HBeAg/IC阳性. 29份中17份(58.7%)为HBeAg/IC阳性的G 1896感染；血清抗HBe阳性组A 1896检出率高于抗HBe阴性组，P<0.05(25% vs 3.2%).
结论 HBeAg/IC为HBV活跃复制指标；临床HBeAg阴性、HBVDNA阳性患者仍多数为G 1896感染，HBeAg/IC形致双抗体夹心法不能检出HBeAg；抗HBe应答可能为促使前C变异的重要因素.
主题词 肝炎，病毒性；肝炎e抗原，乙型；肝炎，乙型
张树林, 朱江, 刘敏, 柴宁莉.血清HBeAg阴性与HBeAg/IC及A1896变异的关系.华人消化杂志,1998;6(11):960-962
0 引言
HBeAg为临床判断HBV复制和传染性的常用血清学标志. 近年发现HBV基因组前C区变异的HBeAg缺陷变异株(最常见为A1896)，无HBeAg产生，血清HBeAg阴性但HBVDNA阳性[1，2]，因此HBeAg检测的临床意义应重新评价. 业已证实，HBV感染者体内可检出e抗原特异免疫复合物(HBeAg/IC)，存在于HBeAg/IC中的HBeAg用现行双抗体夹心法无法检出[3，4]. 本文以单克隆抗-HBe固相ELISA检测血清中HBeAg/IC，以套式多聚酶链反应(n-PCR)检测血清中HBVDNA；3＇碱基特异PCR(3＇-BS-PCR)判断变异HBV(A1896)，探讨HBeAg阴性与HBeAg/IC形成及A1896的关系，评价HBeAg/IC检测的临床意义.
1 对象和方法
1.1 对象 117例慢性HBV感染者系1997-08/1998-04我传染科住院和门诊随访患者，男81例，女36例，年龄17岁～59岁，血清HBsAg阳性，诊断依据1995年北京第五届全国传染病与寄生虫病学术会议制定的《病毒性肝炎防治方案》. 20例正常对照为本校医学生和健康查体者，HBsAg阴性，肝功正常. 所有病例IgM抗-HAV，抗-HCV，抗-HEV阴性.
1.2 方法 采静脉血，分离血清，-20℃保存统一检测.
1.2.1 HBeAg/IC检测(ELISA) 单克隆抗-HBe(3G8细胞株)1∶8000稀释包被，4℃过夜(设阴性、阳性及空白对照)，洗涤后加50μL待检血清，50μLPBS/孔，37℃水浴1h后洗涤，再加HRP标记抗人IgG 100μL/孔，37℃ 水浴1h，洗涤后显色测OD450nm，S/N≥2.1为阳性[5].
1.2.2 血清HBV DNA检测(n-PCR) 引物依HBV基因组S区设计，第二次扩增产物电泳后出现206bp条带为阳性[6].
1.2.3 HBV变异株(A1896)的检测 采用3＇碱基特异PCR(3＇-BS-PCR)，引物序列见表1.
表1 3＇-BS-PCR引物序列
自血清中抽提DNA为模板，Ⅰ+Ⅲ引物扩增阳性为野毒株(G1896)，Ⅱ+Ⅲ扩增阳性为变异株(A1896). 为增加扩增特异性，严格控制退火温度为64℃(经预试验确定). 扩增产物电泳后出现408bp条带为阳性[7，8].
1.2.4 HBsAg,HBeAg,抗-HBe,IgM抗-HAV,抗-HCV，抗-HEV均以ELISA检测.统计学处理 采用卡方检验.
2 结果
2.1 HBeAg/IC 阳性血清中HBVDNA检出率为91.3%(21/23)明显高于HBeAg/IC阴性血清(36.2%，34/94)，P<0.001，见表2.
表2 HBeAg/IC和HBV DNA检出情况比较
χ2=21.7，P<0.001.
2.2 29份HBeAg阴性、HBVDNA阳性血清中，仅5例(17.2%)检出A1896，且其中2例与G1896混合感染伴HBeAg/IC阳性；17例(58.7%)为HBeAg阳性G1896感染，见图1.
A B C D
A：G1896伴HBeAg/IC C：G1896+A1896伴HBeAg/IC
B：G1896无HBeAg/IC D：A1896无HBeAg/IC
图1 HBeAg-/HBV DNA+血清G1896. A1896. HBeAg/IC检出情况.
2.3 抗-HBe阳性 (包括HBeAg/IC阳性)血清中 A1896检出率(25%，6/24)高于抗-HBe阴性血清(3.2%，1/31)，见表3.
表3 抗-HBe与A1896的关系
χ2=3.98，P<0.05.
3 讨论
HBeAg/IC为HBV复制指标. 本文HBeAg阳性血清中HBV DNA检出率(91.3%)显著高于 HBeAg/IC阴性血清(36.2%)，P<0.001. 目前采用双抗体夹心法检测血清中HBeAg， 并常以HBeAg作为病毒复制、传染性的标志. 然而，HBeAg/IC形成可致夹心法不能检出复 合物中的HBeAg，存在局限性[4，5，9]. 本文建立的单克隆抗-HBe固相ELISA方法简单、敏感、特异，可作为临床判断HBV复制的另一个标志，克服HBeAg检测的局限性，尤其适合于无条件查血清HBV DNA而HBeAg又为阴性者.
业已证实，HBV前C区突变可阻断HBeAg合成或分泌，是血清HBeAg阴性、HBV DNA阳性的原因之一[1，2，10]. HBeAg缺陷变异株最常见于核苷酸第1896位点突变(G为A替换)，使第28位色氨酸密码(TGG)转换为终止密码(TAG). 本文采用3＇-碱基特异多聚酶链反应3＇-BS-PCR)确定A1896变异，检测29例HBeAg阴性、HBV DNA阳性血清，结果显示仅5例(17.2%)检出A1896(而且其中2例为与G1896混合感染伴HBeAg/IC阳性)，17例(58.7%)HBeAg/IC阳性，提示临床所见HBeAg阴性，HBV DNA阳性者中多数仍为野毒株(G1896)感染，因HBeAg/IC形成致夹心法查HBeAg阴性，少数为A1896变异株感染.
许多研究认为，HBV前C变异发生与抗-HBe应答密切相关，抗-HBe是选择HBeAg缺陷变异株的条件[11，12]. 本文结果见抗-HBe阳性血清A1896检出率明显高于抗- HBe阴性血清，P<0.05(25.0% vs 3.2%)，证实抗-HBe应答与A1896相关. 抗-HBe出现意味HBeAg特异Th活化，其产生之Th2类细胞因子促B细胞活化产生抗-HBe，Th1类细胞因子诱导CTL活化识别感染细胞表面HBeAg，清除HBV同时导致肝细胞破坏. HBeAg缺陷变异株感染肝细胞表面无HBeAg，逃避免疫攻击而被选择，可见抗-HBe可能为A1896产生的促进因素.
总之，HBeAg/IC可作为HBV复制指标；HBeAg阴性、HBVDNA阳性患者多数仍为野毒株感染，因HBeAg/IC形成致HBeAg无法检出；A1896为HBeAg阴性、HBVDNA阳性的原因之一，但不象是主要原因，A1896出现与抗-HBe应答相关.
4 参考文献
1 Carman WF, Hadziyannis S, Mcgarvey MJ. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis
B infection. Lancet,1989;2(9):588-590
2 Wlus SA, Lok A. Natural occurring hepatitis B core gene mutation. Hepatology,1990;22(1):50-60
3 Maruyama T, Mclanchlan A, Lino S. Serological of chronic hepatitis B infection revisited. J Clin Invest, 1993;91(9):2586-2595
4 Maruyama T, Lin S, Koike K. Serology of acute exacerbation in chronic hepatitis B virus infection.
Gastroenterology, 1993;105(6):1141-1151
5 张树林，仟建林，刘宇卉，狄鹏超. HBe/IC与肝损伤的关系. 中华肝脏病杂志，1988；6(2)：106-107
6 Zhang SL, Han XB, Yue YF. The relation between HBV viremia level of pregnant women and intrauterine infection—nested PCR
for detection of HBV DNA. WJG, 1998;4(1):61-63
7 Gerger B. Comparison of mutation specific PCR and direct sequencing of PCR produets for the detection of the HBV pre C
stop codon. Hepatology, 1992;16(2):102A
8 王小飞，刘华瑞，王锦蓉. 乙型肝炎和肝癌患者乙型肝炎病毒前C区1896位点 突变研究. 中华传染病杂志，1996；14(1)：11-14
9 Maruyama T, Kuwata S, Koike K. Precore wild type DNA and immune complex persist in chronic hepatitis B after seroconversion:
no association between genome conversion and seroconversion. Hepatology, 1998;27(1):245-253
10 Okamoto A, Yotsumoto S, Akahane Y. Hepatitis B viruses with precore region defects prevail in persistently infected hosts
along with seroconversion to the antibody against e antigen. Virology, 1990;64(5):1298-1301
11 田琦琦，骆抗先，陈伟华. HBV前C区变异与HBe转换和慢性肝炎的预后. 中华传染病杂志，1996；14(1)：45-46
12 Xuan Thanh N. Hepatitis may precede the occurence of precore region mutation in HBV genome during infect young carriers.
J Med Virol,1994;44(2):200-205, 百拇医药(张树林 朱江 刘敏 柴宁莉)
项目负责人 张树林，710061，西安医科大学第一临床医学院肝炎研究室.
Correspondence to Shu-Lin Zhang, Hepatitis Lab, First Clinical College, Xi＇an Medical University,Xi＇an 710061, Shaanxi Province, China
收稿日期 1998-07-09
Relation ship between negative serum HBeAg-and formation of HBeAg-specific immune complex and HBV mutant A1896
Shu-Lin Zhang, Jiang Zhu, Min Liu and Ning-Li Chai
Hepatitis Lab, First Clinical College, Xi＇an Medical University, Xi＇an 710061, Shaanxi Province, China
Abstract
AIM To explore the relation between serum HBeAg-negative and HBeAg-specific immune complex (HBeAg/IC)， HBV mutant A1896, to evaluate the clinical significance of HBeAg/IC detetion.
METHODS The serum HBeAg/IC was deteted by ELISA using monolonal antibody against HBeAg. The serum HBV DNA was tested with nested polymerase chain reaction (n-PCR) using nested primers induced from S region of HBV genome. A1896 mutant was determined by 3＇-base specific PCR (3＇-BS-PCR). In this study, 117 patients with chronic HBV infection were investigated. Statistical analysis of data was conducted with X test.
RESULTS The detection rate of serum HBV DNA was greatly higher in sera with positive HBeAg/IC than that in sera with negative HBeAg/IC P<0.001 (91.3% vs 36.2%). Twenty nine patients with positive HBV DNA had no HBeAg in serum, only 5 of them (17.2%) were infected with A1896mutant, but 2 of the 5 patients also had wild type HBV (G1896) and HBeAg/IC in sera, 17 patients (58.7%) were infected with G1896 and had HBeAg/IC in serum. The prevalence of A1896 was much higher in sera with positive anti-HBe than that in sera with negative anti-HBe.
CONCLUSION HBeAg/IC is one of the markers indicating HBV active replication. Most patients with negative HBeAg and positive HBV DNA in serum are infected with wild type HBV (G1896)， the formation of HBeAg/IC results in undetection of HBeAg with “Sandwich” ELISA: Anti-HBe response may be one of the factors that promote the mutation of pre-C gene of HBV.
Subject headings hepatitis, viral; HBeAg; hepatitis B
Zhang SL, Zhu J, Liu M, Chai NL. Relation ship between negative serum HBeAgand formation of HbeAg-specific immune complex and HBV mutant A 1896. Huaren Xiaohua Zazhi,1998;6(11):960-962
摘要
目的 探讨血清HBeAg阴性(双抗体夹心法)与HBeAg/IC形成及HBV变异株A 1896的关系，评价HBeAg/IC检测的临床意义.
方法 单克隆抗HBe固相ELISA检测血清中HBeAg/IC；套式多聚酶链反应检测HBVDNA；3＇碱基特异多聚酶链反应判断A 1896变异；ELISA检测HBeAg、抗HBe，研究对象为117例慢性HBV感染者，20例健康对照统计处理采用卡方检验.
结果 HBeAg/IC阳性血清中HBV DNA检出率明显高于HBeAg/IC阴性血清，P<0.001(91.3% vs 36.2%)；29份HBeAg阴性、HBVDNA阳性血清中仅5例(17.2%)检出A 1896，而且其中2例与野毒株(G 1896)混合感染并伴HBeAg/IC阳性. 29份中17份(58.7%)为HBeAg/IC阳性的G 1896感染；血清抗HBe阳性组A 1896检出率高于抗HBe阴性组，P<0.05(25% vs 3.2%).
结论 HBeAg/IC为HBV活跃复制指标；临床HBeAg阴性、HBVDNA阳性患者仍多数为G 1896感染，HBeAg/IC形致双抗体夹心法不能检出HBeAg；抗HBe应答可能为促使前C变异的重要因素.
主题词 肝炎，病毒性；肝炎e抗原，乙型；肝炎，乙型
张树林, 朱江, 刘敏, 柴宁莉.血清HBeAg阴性与HBeAg/IC及A1896变异的关系.华人消化杂志,1998;6(11):960-962
0 引言
HBeAg为临床判断HBV复制和传染性的常用血清学标志. 近年发现HBV基因组前C区变异的HBeAg缺陷变异株(最常见为A1896)，无HBeAg产生，血清HBeAg阴性但HBVDNA阳性[1，2]，因此HBeAg检测的临床意义应重新评价. 业已证实，HBV感染者体内可检出e抗原特异免疫复合物(HBeAg/IC)，存在于HBeAg/IC中的HBeAg用现行双抗体夹心法无法检出[3，4]. 本文以单克隆抗-HBe固相ELISA检测血清中HBeAg/IC，以套式多聚酶链反应(n-PCR)检测血清中HBVDNA；3＇碱基特异PCR(3＇-BS-PCR)判断变异HBV(A1896)，探讨HBeAg阴性与HBeAg/IC形成及A1896的关系，评价HBeAg/IC检测的临床意义.
1 对象和方法
1.1 对象 117例慢性HBV感染者系1997-08/1998-04我传染科住院和门诊随访患者，男81例，女36例，年龄17岁～59岁，血清HBsAg阳性，诊断依据1995年北京第五届全国传染病与寄生虫病学术会议制定的《病毒性肝炎防治方案》. 20例正常对照为本校医学生和健康查体者，HBsAg阴性，肝功正常. 所有病例IgM抗-HAV，抗-HCV，抗-HEV阴性.
1.2 方法 采静脉血，分离血清，-20℃保存统一检测.
1.2.1 HBeAg/IC检测(ELISA) 单克隆抗-HBe(3G8细胞株)1∶8000稀释包被，4℃过夜(设阴性、阳性及空白对照)，洗涤后加50μL待检血清，50μLPBS/孔，37℃水浴1h后洗涤，再加HRP标记抗人IgG 100μL/孔，37℃ 水浴1h，洗涤后显色测OD450nm，S/N≥2.1为阳性[5].
1.2.2 血清HBV DNA检测(n-PCR) 引物依HBV基因组S区设计，第二次扩增产物电泳后出现206bp条带为阳性[6].
1.2.3 HBV变异株(A1896)的检测 采用3＇碱基特异PCR(3＇-BS-PCR)，引物序列见表1.
表1 3＇-BS-PCR引物序列
| 序号 | 核酸位置 | 序 列 |
| Ⅰ | 1879～1896 | 5＇-GTGCCTTGGGTGGCTTTG-3＇ |
| Ⅱ | 1879～1896 | 5＇-GTGCCTTGGGTGGCTTTA-3＇ |
| Ⅲ | 2270～2287 | 3＇-CACACCTAAGCGTGAGGA-5＇ |
自血清中抽提DNA为模板，Ⅰ+Ⅲ引物扩增阳性为野毒株(G1896)，Ⅱ+Ⅲ扩增阳性为变异株(A1896). 为增加扩增特异性，严格控制退火温度为64℃(经预试验确定). 扩增产物电泳后出现408bp条带为阳性[7，8].
1.2.4 HBsAg,HBeAg,抗-HBe,IgM抗-HAV,抗-HCV，抗-HEV均以ELISA检测.统计学处理 采用卡方检验.
2 结果
2.1 HBeAg/IC 阳性血清中HBVDNA检出率为91.3%(21/23)明显高于HBeAg/IC阴性血清(36.2%，34/94)，P<0.001，见表2.
表2 HBeAg/IC和HBV DNA检出情况比较
| HBeAg/IC | HBV DNA | |
| + | - | |
| + | 21 | 2 |
| - | 34 | 60 |
χ2=21.7，P<0.001.
2.2 29份HBeAg阴性、HBVDNA阳性血清中，仅5例(17.2%)检出A1896，且其中2例与G1896混合感染伴HBeAg/IC阳性；17例(58.7%)为HBeAg阳性G1896感染，见图1.
| 58.7% | 24.1% | 6.9% | 10.3% |
A B C D
A：G1896伴HBeAg/IC C：G1896+A1896伴HBeAg/IC
B：G1896无HBeAg/IC D：A1896无HBeAg/IC
图1 HBeAg-/HBV DNA+血清G1896. A1896. HBeAg/IC检出情况.
2.3 抗-HBe阳性 (包括HBeAg/IC阳性)血清中 A1896检出率(25%，6/24)高于抗-HBe阴性血清(3.2%，1/31)，见表3.
表3 抗-HBe与A1896的关系
| A1896 | 抗-HBe | |
| + | - | |
| + | 6 | 1 |
| - | 18 | 30 |
χ2=3.98，P<0.05.
3 讨论
HBeAg/IC为HBV复制指标. 本文HBeAg阳性血清中HBV DNA检出率(91.3%)显著高于 HBeAg/IC阴性血清(36.2%)，P<0.001. 目前采用双抗体夹心法检测血清中HBeAg， 并常以HBeAg作为病毒复制、传染性的标志. 然而，HBeAg/IC形成可致夹心法不能检出复 合物中的HBeAg，存在局限性[4，5，9]. 本文建立的单克隆抗-HBe固相ELISA方法简单、敏感、特异，可作为临床判断HBV复制的另一个标志，克服HBeAg检测的局限性，尤其适合于无条件查血清HBV DNA而HBeAg又为阴性者.
业已证实，HBV前C区突变可阻断HBeAg合成或分泌，是血清HBeAg阴性、HBV DNA阳性的原因之一[1，2，10]. HBeAg缺陷变异株最常见于核苷酸第1896位点突变(G为A替换)，使第28位色氨酸密码(TGG)转换为终止密码(TAG). 本文采用3＇-碱基特异多聚酶链反应3＇-BS-PCR)确定A1896变异，检测29例HBeAg阴性、HBV DNA阳性血清，结果显示仅5例(17.2%)检出A1896(而且其中2例为与G1896混合感染伴HBeAg/IC阳性)，17例(58.7%)HBeAg/IC阳性，提示临床所见HBeAg阴性，HBV DNA阳性者中多数仍为野毒株(G1896)感染，因HBeAg/IC形成致夹心法查HBeAg阴性，少数为A1896变异株感染.
许多研究认为，HBV前C变异发生与抗-HBe应答密切相关，抗-HBe是选择HBeAg缺陷变异株的条件[11，12]. 本文结果见抗-HBe阳性血清A1896检出率明显高于抗- HBe阴性血清，P<0.05(25.0% vs 3.2%)，证实抗-HBe应答与A1896相关. 抗-HBe出现意味HBeAg特异Th活化，其产生之Th2类细胞因子促B细胞活化产生抗-HBe，Th1类细胞因子诱导CTL活化识别感染细胞表面HBeAg，清除HBV同时导致肝细胞破坏. HBeAg缺陷变异株感染肝细胞表面无HBeAg，逃避免疫攻击而被选择，可见抗-HBe可能为A1896产生的促进因素.
总之，HBeAg/IC可作为HBV复制指标；HBeAg阴性、HBVDNA阳性患者多数仍为野毒株感染，因HBeAg/IC形成致HBeAg无法检出；A1896为HBeAg阴性、HBVDNA阳性的原因之一，但不象是主要原因，A1896出现与抗-HBe应答相关.
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