外周血单个核细胞中乙型肝炎病毒前S／S基因变异？…

目的 研究外周血单个核细胞（ＰＢＭＣ）中乙型肝炎病毒（ＨＢＶ）包膜抗原基因变异的意义。方法 收集１９例慢性乙肝患者的ＰＢＭＣ和配对血清,用ＰＣＲ扩增ＰＢＭＣ及血清中ＨＢＶ包膜基因片段９Ｓ,ｐｒｅＳ１,ｐｒｅＳ２）,并对其中各５例ＰＢＭＣ及配对血清中的ｐｒｅＳ１和ｐｒｅＳ２基因片段,分别进行核苷酸序列分析。结果 Ｓ,ｐｒｅＳ１和ｐｒｅＳ２片段在血清和ＰＢＭＣ中的检出率,分别为１００％,９４．＆％,１     

我国急性散发性戊型肝炎病毒部分核苷酸序列分析

用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）自深圳、长春、杭州等地４１份急性散发性戊型肝炎病人血清中获得２８株ＨＥＶｃＤＮＡ，对其中３株ＨＥＶｃＤＮＡ的ＯＲＦ２基因片段，用荧光法直接测定其核苷酸序列，并与戊型肝炎病毒墨西哥株（Ｍ）、缅甸株（Ｂ）和新疆流行株（ＣＨ１·１）进行了比较，结果该３株散发性ＨＥＶ与Ｍ株的核苷酸序列同源性分别为８０．２％、７９．９％和７９．４％；与Ｂ流行株的同源性为９５．５％、９３．９％和９５．１％；与散发株的同源性为９３．４％、９２．３％和９３．８％；与ＣＨ１·１的同源性为９７．０％、９６．５％和９５．９；表明该３株散发性ＨＥＶ与ＨＥＶ（Ｂ）和ＣＨ１·１的核酸序列同源性较高，可能属同一亚型。     

慢性乙型肝炎表面抗原携带者前S1/S2基因的相似株现象

为了解慢性ＨＢＶ携带者体内ＨＢＶ病毒的状况，以选择ＨＢＶ前Ｓ１／Ｓ２为研究区段，应用半巢式ＰＣＲ法从３例慢性ＨＢＶ携带者血清中扩增出ＨＢＶ前Ｓ１／Ｓ２基因，分别将其克隆于噬菌体Ｍ１３ｍｐ１８、Ｍ１３ｍｐ１９，每例随机筛选１０个阳性克隆，进行序列分析及遗传距离分析，结果发现：３例病人各克隆分别与同源性最好的ＨＢＶ野生株相比，存在着明显的碱基替代、缺失和插入。此类病人体内的ＨＢＶ株同源性差，克隆间最大的遗传距离为０．１２５。慢性ＨＢＶ携带者体内的ＨＢＶ株呈“相似株”分布。     

乙型肝炎病毒pre S基因真核表达质粒诱导小鼠特异性？…

本文构建了含有ＨＢｓＡｇ ｐｒｅ Ｓ区基因的真核表达载体ｐＣＭＶ４－ｐｒｅ Ｓ，经大量提取质粒并纯化后，肌肉注射Ｂａｌｂ／ｃ小鼠，共免疫３次，间隔两周，结果有３／７的小鼠血清抗ｐｒｅ Ｓ２抗体呈阳性。     

乙型肝炎病毒核心基因的酶切图谱及核苷酸序列分析

对慢性乙型肝炎患者的２２份肝穿刺活检组织及其中配对的６份血清用ＰＣＲ扩增出乙型肝炎病毒（ＨＢＶ）的ＰｒｅＣ／Ｃ基因，选用ＡｖａⅡ、Ｓａｕ３ＡⅠ、ＸｍｎＩ，ＢｓｔＮＩ及ＴａｑＩ５种限制性内切酶消化后，对Ｃ基因进行酶谱分析。发现有６份肝组织及１份血清出现异常酶谱。对Ｓａｕ３ＡⅠ酶解异常标本进行分子克隆及核苷酸序列分析，发现２例患者因２１３７位核苷酸点突变出现了Ｓａｕ３ＡⅠ的新酶切点。类似变化在肝癌组织的ＨＢＶＣ基因中也曾发现，其意义待进一步研究。     

乙型肝炎病毒pre S基因真核表达质粒诱导小鼠特异性抗体反应的研究

本文构建了含有ＨＢｓＡｇ ｐｒｅ Ｓ区基因的真核表达载体ｐＣＭＶ４ｐｒｅ Ｓ, 经大量提取质粒并纯化后, 肌肉注射Ｂａｌｂ／ｃ 小鼠, 共免疫３ 次, 间隔两周, 结果有３／７ 的小鼠血清抗ｐｒｅ Ｓ２ 抗体呈阳性。     

云南省陇川县HIV—1流行毒株膜蛋白基因C2—V3区序列测定…

使用ＰＣＲ对２１份采集于１９９５年中的云南陇川县ＨＩＶ－１阳性静脉吸毒者外周血单个核细胞（ＰＢＭＣｓ）样品进行扩增，从１７份样品中获得了ＨＩＶ－１膜蛋白（ｅｎｖ）基因的核酸片段，并对其Ｃ２－Ｖ３及邻区４５０个核苷酸序列进行了测定和分析。研究结果表明，１７份陇川样品中存在Ｂ和Ｃ两种亚型的ＨＩＶ－１毒株序列，各亚型内的基因离散率分别为４．７％和３．３％。与Ａ￣Ｅ参考亚型及部位Ｂ和Ｃ亚型代表株序列相比较     

IL—2／HBV PreS融合基因的克隆及序列测定

目的：获得ＩＬ－２／ＨＢＶｐｒｅＳ融合基因克隆，为表达ＩＬ－２／ＨＢＶｐｒｅＳ融合蛋白奠定基础，方法：用ＰＣＲ方法从乙肝全序列中扩增ＨＢＶｐｒｅＳ基因片段，并克隆入带ＩＬ－２基因的ｐｌｙ５质粒中，挑出的阳性克隆再亚克隆到ｐＵＣ１８中进行序列测定。结果：基因全长９３０ｂｐ，编码３１０个氨基酸，序列内有起始码和终止码。结论：所克隆的基因为ＩＬ－２与ＨＢＶｐｒｅＳ的融合基因。     

中国人庚型肝炎病毒E1区5＇端基因相关多肽的抗原性初步研究

用逆转录－ＤＮＡ聚合酶链式反应（ＲＴ－ＰＣＲ）从１例中国庚型肝炎（ＨＧＶ）患者血清中扩增出含Ｅ１前区部分基因及Ｅ１区５＇端共３０９ｈｐ基因片段。对其中包括Ｅ１区９６ｂｐ在内的１２０ｂｐ核苷酸进行了序列分析，发现此区段基因与录入号为Ｕ４４４０２Ｇｅｎｅｂａｎｋ报道的美国发现的ＨＧＶ相关序列具有９３％的同源性，氨基酸的同源性为９８％。Ｇｏｌｄｋｅｙ蛋白质分析程序显示，此区段的Ｅ１区可能存在抗原位点。化学合成可能为抗原位点长约２９个氨基酸的多肽Ｅ１Ｐ１。利用Ｅ１Ｐ１及ＮＳ３区内短肽ＮＳ３Ｐ１包被的ＥＬＩＳＡ方法检测了１２名非甲－戊肝炎患者血清。Ｅ１Ｐ１检出的２份血清（２／１２）中有抗－Ｅ１Ｐ１ＩｇＧ的存在。ＮＳ３Ｐ１捡出的３份血清（３／１２）中有抗－ＮＳ３Ｐ１ＩｇＧ的存在，其中包括Ｅ１Ｐ１检出的两份阳性血清。本结果说明在ＨＧＶＥ１区内至少有一个抗原位点存在。     

乙型肝炎病毒S基因129Leu和145Arg变异株的抗原表达？…

目的 研究我国发现的乙型病毒Ｓ基因突变株表达ＨＢｓＡｇ及ＤＮＡ免疫的特性。方法 用自乙肝疫苗免疫无保护婴儿血清中克隆的２株变异Ｓ基因片段,置换已知核苷酸序列并可表达及复制的ＨＢＶ１．２个拷贝全基因野毒株重组质粒Ｐ３．８Ⅱ的Ｓ基因,将重组质粒转染ＨｅｐＧ２细胞,用Ａｂｂｏｔｔ试剂和２４种单抗检测染细胞培养上清中ＨＢｓＡｇ的结合力。用重组质粒ＨｅｐＧ２细胞,用Ａｂｂｏｔｔ试剂和２４种单抗检测转染细胞培     

HBV与HCV融合DNA疫苗的构建及其体液免疫应答

目的 构建含乙型肝炎病毒（HBV）表面抗原基因（S区基因）与丙型肝炎病毒（HCV）核心抗原基因（C区基因）的嵌合真核表达载体,观察preS1和preS2基因对HBV表面抗原及HCV核心抗原体液免疫的影响。方法 用PCR方法,分别扩增HBV S区基因和HCV C区基因。将S区基因克隆入真核表达载体pcDNA3.1,酶切鉴定后,大量提取质粒并免疫Balb/c小鼠,用ELISA法检测抗HBs和抗HCV抗体。结果 成功地扩增出目的基因片段,克隆后酶切鉴定结果正确,序列分析与文献报告相一致。免疫后检测到抗HBs和抗HCV抗体。preS1与preS2基因对构建的融合DNA疫苗的体液免疫应答有一定的抑制作用。抗HBs抗体的产生低于只含S基因的真核表达载体;preS1基因对抗HCV抗体的产生具有抑制作用,而preS2无影响。结论 不同长度的HBV S区基因可影响抗HBs和抗HCV抗体的产生。     

Hepatitis B defective virus with rearrangements in the preS gene during chronic HBV infection.

We have found a defective form of HBV2 in a HBsAg- and anti-HBe-positive patient with liver cancer. Viral deletions were identified in the preS coding region using PCR. The presence of deleted HBV forms was observed in serum, PBMC, and liver samples. After sequencing 12 clones were analyzed (subtype adr). In 9 out of 12 clones a 183-bp in-frame deletion was recorded in the preS1 region (2995 to 3177). Three out of 9 clones also yielded rearrangements of the preS2 N-terminal part. Four out of 9 showed numerous point mutations in the preS1 and preS2 sequence. In addition, 3 out of 12 clones, which did not show the 183-bp preS1 deletion were found to have small deletions and insertions in the same part of the preS1 gene. Immunological mapping using monoclonal anti-preS antibodies showed loss of preS epitopes located at the 3'-part of preS1 and the 5'-part of preS2. On the other hand, epitopes mapped to the 5'-part of preS1 and 3' of preS2 were conserved. PBMC were also tested and solely PCR showed the major form of defective HBV with preS1 183-bp deletion. However, viral deletions in the preS gene eliminated the preS2 promotor region and B- and T-cell recognition sites. In contrast to this, the preS1 binding site to hepatocytes was conserved. Therefore, such deletions would potentially lead to an impairment in viral clearance without affecting viral penetration in liver cells, possibly accounting for chronic HBV infection.     

乙肝疫苗母-婴阻断失败者病毒全基因变异分析

目的 探讨乙肝疫苗母-婴阻断失败者中乙肝病毒(HBV)全基因变异状况.方法 用PCR方法对启东地区11对乙肝疫苗免疫失败儿童-母亲配对血清及6例疫苗接种成功儿童的"大三阳"母亲血清扩增HBV全长基因,克隆测序后以Clustal X软件进行序列比对.病毒载量采用实时荧光定量PCR方法测定.结果 疫苗阻断失败和成功儿童的母亲HBV平均滴度分别为(1.2×107±3.1×1010)copies/ml和(1.6×107±8.8×1010)copies/ml,两组之间差异无统计学意义(P>0.05).在11例HBV DNA阳性的儿童中,4例(36.4%)有HBsAg"a"决定簇的氨基酸改变,表现为T125A、I126T、Q129H、M133V、D144V和G145A等6种不同突变形式,但母亲中HBsAg"a"决定簇均为野生型.疫苗接种失败的儿童中HBV preS1、preS2、S、X、preC/C和P基因的平均突变率分别为0.38%、0.22%、0.27%、0.17%、0.11%和0.11%.preS1区nt2999-3157、S区nt529-677、C区nt1955-2016和P区nt923-1001、nt2489-2602为病毒基因组中最易发生突变的区域.结论 经主动和被动免疫后,儿童体内HBV突变可发生于所有开放阅读框架内.除s基因外,preS和P基因的突变可能也与免疫逃逸有关.     

PreS deletion mutations of hepatitis B virus in chronically infected patients with simultaneous seropositivity for hepatitis‐B surface antigen and anti‐HBS antibodies

Hepatitis B surface antigen (HBsAg) and anti‐HBs antibodies (anti‐HBs) may coexist in certain chronic hepatitis B (CHB) patients. This study was designed to further explore the relationship between this coexistence and hepatitis B Virus (HBV) preS deletions. Sera of 28 patients carrying both HBsAg and anti‐HBs (Group I) and those of another 28 HBsAg positive but anti‐HBs negative patients (Group II) were collected from CHB patients. Direct sequencing of polymerase chain reaction products or sequencing of clones was applied to both groups to determine sequences of HBV preS and S genes. Genotyping of the S gene indicated that all sampled HBVs were either Genosubtype Ba or Genosubtype Ce. Seven samples in Group I harbored HBV preS deletion mutations. Three of the seven samples showed large deletion mutations in 3′ terminus of preS1 and co‐existence of the mutant type and the full‐length wild type, and the remaining four samples showed deletion mutations in 5′ terminus of preS2. All mutant strains were found to be genosubtype Ce. Only two samples in Group I showed G145R/A mutation. Only one sample in Group II contained preS deletion mutation. It is therefore concluded that HBV preS deletion mutations are likely to be related to the coexistence of HBsAg and anti‐HBs in CHB patients (P‐value = 0.024). Some immune reactions may select for the preS deletion in CHB patients with anti‐HBs, the possible marker for immune selection. J. Med. Virol. 82:23–31, 2010. © 2009 Wiley‐Liss, Inc.     

反基因锁核酸体外抑制乙肝病毒前S1基因表达

 目的 探讨针对乙肝病毒前S1基因同聚嘌呤区的锁核酸体外抑制细胞内病毒复制的作用。方法 针对乙肝病毒前S1基因同聚嘌呤区,利用RNAstructure软件分别设计合成锁核酸、硫代寡核苷酸、未修饰寡核苷酸及无关对照序列,以阳离子脂质体介导转染HepG2.2.15细胞,采用荧光定量聚合酶链反应技术（FQ-PCR）、时间分辨免疫荧光技术（TRFIA）和酶联免疫法（ELISA）分别监测1、3、5和7 d细胞培养上清液中HBV DNA、HBsAg和前S1抗原的含量;四甲基偶氮唑蓝（MTT）法检测锁核酸对细胞代谢的影响。结果 加入锁核酸后,对HBV DNA复制、HBsAg和前S1抗原表达均显示有较强的抑制作用,且抑制率随时间呈增高趋势,7 d后抑制率分别达64.32%、67.51%和63.88%。LNA对细胞代谢无明显影响。结论 针对乙肝病毒前S1基因同聚嘌呤区的反基因锁核酸,体外能有效抑制乙肝病毒的复制,既为乙肝病毒治疗提供有效靶位,也为反基因治疗提供理论和实验依据。     

The relation between virus genotypes and coexisting surface antigen and antibody in patients with hepatitis B, and the development of preS region deletions

In recent years, there has been renewed interest in hepatitis B virus (HBV) genotypes. Our previous data have shown the importance of in-frame deletions in the preS region in cases of coexisting hepatitis B surface antigen (HBsAg) and anti-hepatitis B surface antibody (HBsAb). The aim of the present study was to investigate the relation between HBV genotypes and coexisting HBsAg and HBsAb, preS deletion mutants. We investigated the HBV genotypes in 9 patients with coexisting HBsAg and HBsAb. Viral DNA was extracted from the patients' sera and the HBV S gene region was amplified by polymerase chain reaction (PCR). HBV genotypes were then investigated by restriction fragment length polymorphism(RFLP) analysis. All 9 cases were found to have genotype C. This result clearly indicates that the unique finding of coexisting HBsAg and HBsAb depends on the HBV genotype. After genotypic screening was performed for HBV-positive samples from randomly selected 60 cases. The results of the 60 cases we investigated showed 26 cases of genotype B (43.3%), 31 cases of genotype C (51.7%), 1 case of coexisting genotype B and C (1.7%), and 2 cases of other genotypes (3.3%). Of the 60 cases, 45 cases consisting of 21 with genotype B and 24 with genotype C were subject to direct DNA sequencing of PCR products in the preS region to determine the presence or absence of preS deletion mutants. PreS deletion mutants were found in a total of 7 of the 45 HBV cases that underwent sequencing(7/45; 15.6%), and 6 of these had genotype C (6/24 cases, 25.0%), whereas only 1 had genotype B (1/21 cases, 4.8%). These results demonstrate a greater frequency of preS deletion mutants with genotype C. Interestingly, many preS deletion mutants showed deletions at the same point, namely the amino terminal side of the preS2 region. These results indicate that the HBV genotype is involved in the molecular pathogenesis of hepatitis B.     

HBsAg阴性preS1Ag阳性的HBV携带者HBVS基因多态性分析

目的探讨HBsAg（-）／HBeAg（＋）／HBcAb（＋）／preS1Ag（＋）少见血清学模式形成的原因。方法采用聚合酶链反应（PCR）、单链构象多态性分析（SSCP）、异源双链分析（HA）和基因测序的方法，对该少见模式乙肝病毒携带者HBVS基因进行分析，并与其母亲及参考序列HBVS基因进行比较，分析其变异情况。结果该少见模式乙肝病毒携带者与其母亲HBVS基因PCR扩增结果均为阳性，经SSCP、HA和核苷酸序列比对分析，两者HBVS基因多态性不明显，同源性为99．82％。与其母亲或参考序列（AF411409）比对，该少见模式乙肝病毒携带者HBVS基因在第353位由野生型的C变为A，从而使HBsAg118位氨基酸由苏氨酸变为赖氨酸（aa118T→K）。结论该少见模式乙肝病毒携带者所感染的HBV在HBsAg第118位氨基酸上错义突变（T→K），改变了HBsAg的空间结构和抗原性，影响了HBsAg与抗-HBs的结合能力。