动态分析老年重症乙肝患者乙肝病毒全基因组及其表达蛋白抗原性变化

目的 动态分析1例老年重症乙型肝炎患者全基因组序列变化，在全基因组水平上阐述HBV基因组变异对其不同基因生物学特性的影响。方法 一次扩增全长乙型肝炎病毒(hepatitis B virus，HBV)DNA基因组，测序鉴定，建立HBV全基因组克隆、转染表达。结果 通过测序鉴定、同源性比较和真核细胞转染分析，显示HBV基因组序列变异集中在S基因，其表达蛋白前后有抗原性变化。结论 S基因变异可以导致S蛋白抗原性变化，打破宿主体内免疫耐受，诱发重症肝炎的发生。     

三种鸭乙肝病毒感染情况的研究

DHBV的感染与肝病的关系还不甚明了。本文用斑点杂交法,液闪法和光镜病理分别对92只从市场采购的家鸭进行研究发现:A组(绍兴麻鸭)46只中有28只DHBVDNA(+)(60.8%),B组(昆山麻鸭)18只中有7只DHBV DNA(+)(38.9%),二组比较(P>0.05)无显著性差异,C组(天津白鸭)28只无一只DHBV DNA阳性,说明南方鸭比天津鸭易感。同时用DHBVDNA探针与人HBV DNA阳性的血清行杂交和用HBVDNA探针与鸭DHBV DNA阳性的血清行杂交结果均为阴性,说明二者之基因序列无同源性。DNA聚合酶(DNA-p)的检测发现其阳性率为6.5%并同时伴有DHBV DNA(+),说明DNA-p的检测     

乙型肝炎病毒全长基因的扩增及克隆

目的 建立血清标本经聚合酶链反应(PCR)扩增乙型肝炎病毒(HBV)全长基因的新方法,并初步进行克隆分析,从而为HBV分子生物学及致病机理研究奠定基础.方法 针对位于整个HBV基因序列中的两个缺口区设计含酶切位点的引物,扩增3.2 kb HBV全长DNA,经酶切及连接后克隆人PUC18载体.结果 用PCR方法成功获得了3.2 kb HBV DNA,经Sal Ⅰ酶切克隆入PUC18载体,获得重组质粒PUC18/HBV3.2,酶切鉴定和PCR扩增证实重组质粒中含有3.2 kb HBV全长DNA,成功构建了HBV全基因克隆.结论 成功建立了从患者血清中克隆HBV全基因组的方法,为从全基因水平深入研究乙型肝炎病毒变异与致病机理的关系奠定了基础.     

乙型肝炎病毒前-S1蛋白结合蛋白小鼠同源基因的生物信息学分析

目的克隆、鉴定乙型肝炎病毒(HBV)前S1蛋白结合蛋白(PS1BP)小鼠同源基因,并进行初步的生物信息学分析。方法以克隆的人PS1BP基因序列作为参照,利用美国国立卫生研究院国立医学图书馆生物工程信启、学中心建立的的核苷酸数据库,以及在线核苷酸序列同源性比对分析软件BLASTn,与数据库中已经收录的小鼠cDNA序列进行比对,确定PS1BP小鼠同源基因,比较人和小鼠PS1BP的蛋白质一级结构序列的同源性。对小鼠PS1BP蛋白质一级结构的特点进行生物信息学分析,并确定小鼠PS1BP基因组DNA的结构特点。结果通过不同种属核营酸序列的同源性比对,确定小鼠PS1BP的cDNA由1455nt组成,编码产物由484aa组成。与人PS1BP蛋白质一级结构的同源性为84．92％(411／484)。对小鼠PS1BP蛋白一级结构序列进行生物信启、学分析,发现一系列潜在的蛋白修饰位点。以小鼠PS1BP的cDNA序列作为参照,对高通量基因序列数据库进行例源基因的搜索,发现小鼠的一段基因组DNA序列(AC117576．3)与之同源,进一步的分子结果表明,小鼠的PS1BP基因组DNA含有3段外显子序列,2段内含子序列。结论确定了小鼠PS1BP基因序列,为进一步研究其功能和生物学作用奠定了基础。     

突变乙型肝炎病毒核心蛋白的克隆及表达

目的 克隆并表达发生长片段缺失的HBV核心蛋白(HBV-C)基因,并对其进行DNA序列、蛋白质结构和抗原性分析.方法 采用PCR方法从1株野生型HBV基因组中扩增得到发生长片段缺失的HBV-C基因,克隆至pUCm-T质粒,进行测序、同源性比较和蛋白质结构分析;再将基因编码区克隆至原核表达载体pET-28a,构建含HBV-C基因的重组表达质粒,转化至大肠埃希菌BL21中进行诱导表达并检测其抗原性.结果 PCR扩增出的HBV-C基因长度经序列分析表明,其核苷酸序列缺失了220 bp至317 bp之间的98个碱基,造成从第74个氨基酸起发生移码突变并失去了抗原性.结论 成功克隆和表达了发生长片段缺失的HBV-C基因.     

人肝再生增强因子基因组DNA的克隆化与序列分析

目的克隆人肝再生增强因子(ALR)的基因组DNA,并进行序列分析。方法采用人肝再生增强因子cDNA及其编码序列,对基因的核苷酸序列数据库(GenBank)以BLAST为工具进行核苷酸序列同源性比较分析,寻找相应的ALR基因组DNA序列。结果从GenBank核苷酸序列数据库中寻找到人ALR基因组DNA全序列,由1813个核苷酸组成。人ALR基因组DNA序列由3段外显示子(exon)组成,分别为18nt,197nt和163nt。基因的编码产物由125个氨基酸残基组成,与小鼠ALR基因组DNA结构类似。基因组DNA定位于染色体的16p13.3位点上。结论克隆了人ALR基因组DNA全序列。     

乙型肝炎病毒表面抗原基因的克隆及序列分析

目的克隆并鉴定乙型肝炎病毒表面抗原基因,为下一步构建该重组腺病毒载体以及进一步研究腺病毒载体的包装及在乙型肝炎病毒基因治疗中的作用。方法参照人 HBV adr 亚型序列,设计和合成 S 基因特异引物。应用 PCR 技术,从含有 HBsAg 的 HBV DNA 中扩增目的 DNA,通过 TA 连接将其克隆人 pGEM-T easy 载体,经限制性内切酶 BglⅡ/SalⅠ鉴定后,进一步测序鉴定。结果从乙肝表面抗原阳性(HBsAg )血清中成功提取 HBV DNA,并以此 DNA 为模板,成功扩增出 S 基因,测序结果与 GenBank 中注册的相应序列比对,核苷酸序列的同源性高达92％～99％,预测氨基酸序列同源性亦达82％。结论从 HBsAg 阳性血清中成功克隆出 S 基因序列,为进一步构建重组腺病毒及后续实验奠定了基础。     

小分子乙型肝炎病毒基因组缺失突变体结构分析

目的 了解小分子HBV基因组缺失突变体的基因结构及特点.方法 采用一步法PCR从慢性乙型肝炎患者血清中扩增全基因组HBV DNA,回收并克隆＜1 kh的小分子HBV DNA,测序并以BioEdit及VectorNTI 6.0软件分析基因结构特点.结果 获得基因长度介于174～986 bp之间的64种、共124个小分子HBV缺失突变体DNA,按基因结构特点分为3类,即3种GT-AG剪接变异体、29种"常规"缺失突变体及32种基因组内部含polyA的缺失突变体.所有小分子基因组缺失突变体在HBV各编码区及基因调控区均存在不同程度缺失,其中66%(42/64种)保留与HBV复制(包装)相关的所有顺式调控序列,48%(31/64种)保留X编码区.结论 乙型肝炎患者血清中普遍存在小分子HBV基因组缺失突变体,深入了解这些变异体的结构和功能,有助于进一步探索HBV的致病机制.     

一株新疆维吾尔族D型乙型肝炎病毒的全基因克隆分析

目的 进行一株新疆维吾尔族D型HBV的全基因克隆.方法应用PCR扩增HBV全基因,进行全基因克隆,挑取阳性克隆进行序列测定,使用BioEdit和美国国立生物技术信息中心(NCBI)-BLAST工具进行生物信息学分析.结果获得1例新疆维吾尔族慢性HBV感染者D型HBV病毒株的全基因序列,其基因组全长为3174 bp,与目前D基因型参照序列比较,同源性为92%～98%,与1株来自中国的DC重组基因型(AY862865)比较,同源性为91%.进一步分析发现,该新疆病毒株与目前D基因型基因库参照序列比较,有9个碱基缺失,位于核苷酸第1760～1768位(ATTAAAGGT),即可能发生了缺失突变.氨基酸分析发现,其血清型为ayw2型.重组位点分析发现,该新疆病毒株未与其他基因型重组.该新疆株与2株来自土耳其的HBV株(AY796032和AY721605)遗传距离最近.结论成功克隆了一例维吾尔族慢性HBV感染者D型HBV病毒株的全基因序列,新疆维吾尔族D基因型HBV病毒株有其自身特点.     

乙型肝炎病毒基因组结构与功能复杂性的认识

全世界有乙型肝炎病毒(HBV)感染者3.5亿人.对于HBV多年的研究已经积累了丰富的资料,但是,我们对于HBV基因组特性的认识远远没有穷尽,事实上还有许多方面需要进行探索.通过对特定慢性HBV感染者血清中不同的HBV病毒基因克隆序列的比较提出了HBV准种特点,使我们对于HBV基因组结构与功能的复杂性有了更为深入的了解.野生型病毒之间、野生型病毒与突变型病毒之间、突变型病毒之间的反式调节机制是各种类型的缺陷型HBV存在的重要条件和机制,也是HBV感染引起肝细胞癌(HCC)的重要的分子生物学机制.外周血中存在羧基末端截短的表面抗原中蛋白(MHBst)的编码基因,使我们对于HBV基因编码的反式激活蛋白的类型有了新的认识.通过对克隆的HBVDNA全长基因序列的比较,以及与其他地域所流行的HBV DNA序列之间的比较,发现了新型的开放读码框架(ORF),如前-X(pre-X)蛋白的编码基因和前-前-S(per-pre-S)蛋白的编码基因.长距离精确聚合酶链反应(LA-PCR)技术克隆的HBV DNA全长序列,是我国流行的adr亚型的HBVDNA全基因序列,代表了真正存在的HBV的基因全长序列,在HBV基因结构与功能的研究中,以及在抗HBV治疗疗效应答的研究中具有重要的理论和实际应用价值.     

广西麻鸭乙型肝炎感染病毒动物模型的实验研究

目的探讨广西麻鸭作为乙型肝炎病毒感染动物模型的可能性。方法应用广西1 d龄雏麻鸭经腹腔感染鸭乙型肝炎病毒(DHBV),13 d后采用实时荧光定量PCR法检测麻鸭血清DHBV含量,筛选出DHBV强阳性鸭。结果共检测148份麻鸭血清标本,其中DHBV阳性标本136份,阳性率为91.9%。结论广西麻鸭可作为乙型肝炎病毒感染动物模型。实时荧光定量PCR法检测DHBV敏感性较高,重复性好,可用于DHBV检测。     

Naturally occurring infection of Pekin duck embryos by duck hepatitis B virus.

We tested the hypothesis that duck hepatitis B virus (DHBV) is a naturally occurring congenital infection of Pekin duck embryos. Of 219 embryos, 5-25 days after being laid, sera from 30 were found to be positive for endogenous DNA polymerase activity characteristic of hepatitis B-related viruses. The presence of the duck virus was confirmed by hybridization with cloned DHBV DNA. Viral DNA was also found in the livers of embryos incubated for 12 or 18 days. Electrophoretically different forms of DHBV DNA were identified in liver extracts that were not present in serum. These additional liver forms probably represent viral replication intermediates. These observations suggest that the vertical route is a major pathway of DHBV transmission and that viral replication may be initiated by the 12th day of embryonic life.     

Inflammation of the liver causes mutations in duck hepatitis B virus genome

To investigate whether hepatitis causes mutation in the viral genome, DNA sequences in the pre-core region of duck hepatitis B virus (DHBV) DNA were analyzed in both ducks with hepatitis and without hepatitis. Five DHBV carrier ducks were injected with DHBV particle proteins purified from duck serum with Freund’s complete adjuvant (FCA) intrahepatically from 14 day posthatch for 9 weeks (immunized group). Serum was drawn at the end of the 1st and 4th week after the 1st injection of DHBV particle protein and ducks were killed at the end of the 9th week to obtain the liver. Another five ducks without treatment were used as controls. All ducks of the immunized group showed moderate to severe hepatitis at the 9th week. All ducks in the immunized group showed one mutation except one duck that showed two mutations only at the 9th week. Mutations were observed in the 5th, 13th, 21st, 22nd, and 28th codon of the precore region. All of them were point mutation at the 3rd base in the triplets. The frequency of mutation was different in each duck from 20% to 60% but not 100%. There was no mutations in ducks in control group. These results suggest that hepatitis causes mutation in the pre-core lesion genome of duck hepatitis B virus.     

拉米夫定联合泛昔洛韦抗鸭乙型肝炎病毒的实验研究

观察核苷酸类似物拉米夫定联合泛昔洛韦体内抗鸭乙型肝炎病毒（DHBV）的作用。方法 采用重庆麻鸭乙型肝炎动物模型,用拉米夫定联合泛昔洛韦口服治疗4周,停药观察1周,检测用药前后血清中的DHBV DNA、DHBsAg及血清转氨酶（ALT、AST）、肝组织HE染色病理。并以单用拉米夫定、泛昔洛韦、阿昔洛韦作对照。结果 拉米夫定联合泛昔洛韦用药后能使血清中DHBV DNA含量总体水平显著降低（P＜0．01）,停药1周后DHBV DNA较用药4周时DHBV DNA含量回升现象不明显。用药前后清血DHBsAg的吸光度值（490nm)的变化与DNA含量改变相似;此外,肝脏病理检查及治疗4周、停药1周后血清转氨酶检测未发现联合用药对鸭肝组织有明显的毒性损害。结论 拉米夫定联合泛昔洛韦连续用药4周在鸭体内有抗鸭乙型肝炎病毒的作用,且停药后DHBV DNA无明显“反跳”,二者用药有协同作用。     

The relationship between duck hepatitis B virus infection and duck liver diseases in Qitong, China

The relationship between duck hepatitis B virus (DHBV) infection and duck liver diseases was analysed by spot and gel blot hybridization in sera and liver tissues. One hundred and forty ducks were obtained from Qitong county in China. The DHBV-infected rate was 65.7% and the incidence of duck liver diseases was 70%. The state of DHBV DNA was free in the hepatocytes. The detection rate of DHBV DNA in liver was higher than in serum. The results showed that Qitong duck liver diseases are closely correlated to DHBV infection. The incidence of Qitong duck liver diseases and the DHBV infection rate were different in various species of ducks. Qitong ducks have several hepatic pathological changes as seen in human beings. Therefore, Qitong ducks are most appropriate as an experimental model for human HBV infection.     

Enhanced duck hepatitis B virus gene expression following aflatoxin B1 exposure

Epidemiological studies have suggested synergistic interactions between chronic hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure in the etiology of hepatocellular carcinoma (HCC), although the molecular mechanisms of their interactions are still not understood. The aim of this study was to use the Pekin duck model to investigate the impact of AFB1 exposure on duck hepatitis B virus (DHBV) replication during the early stages of virus-carcinogen interactions. Six-week-old chronic DHBV-carrier or uninfected ducks were exposed to AFB1 for 5 weeks or treated with dimethylsulfoxide (DMSO) as a control. Animals were observed for 6 to 13 weeks after AFB1 treatment to study the influence of AFB1 exposure on DHBV replication and liver pathologies. Histological analysis showed more marked changes in the livers of AFB1-treated ducks, and these were enhanced by DHBV infection. A significant increase in serum and liver DHBV DNA level was observed in AFB1-treated ducks as compared with DMSO-treated controls. In addition, viral RNAs, in particular the pregenomic RNA that is the template of viral replication, and intrahepatic DHBV DNA replicative intermediates, were significantly increased by AFB1 treatment. Moreover, an overexpression and accumulation of DHBV large envelope (L) protein was observed in the hepatocytes of AFB1-exposed animals. The in vitro study has further confirmed an increase in intracellular viral DNA and in virus release in AFB1-treated primary duck hepatocytes. Taken together, our results indicate that AFB1 exposure leads to an increase in virus gene expression associated with intrahepatic accumulation of DHBV L protein and enhanced liver pathology.     

肝苏颗粒浸膏粉抗鸭乙型肝炎病毒的实验研究

目的：观察肝苏颗粒浸膏粉（以下简称肝苏）体内抗鸭乙型肝炎病毒（DHBV）的作用,为临床治疗乙肝病毒感染提供实验依据,方法：采用重庆鸭乙型肝炎动物模型,用肝苏口服治疗4W,停药观察1W,检测用药前后血清的DHBVDNA,DHBsAg,转氨酶（ALT,AST）及肝组织病理变化,结果：肝苏各剂量组用药后血清DHBV DNA滴度显著降低或极显著降低（P＜0．05或P＜0．01）,停药1W后中,小剂量组血清DHBV DNA有明显回升现象,而大剂量组DHBV DNA回升现象不明显,其抗病毒效果与剂量大小有一定的关系,用药前后血清DHBsAg O,D值（490nm)的变化与DNA滴度改变相似,此外,肝脏病理学检查及治疗4W,停药1W后血清ALT,AST检查未发现该药对鸭肝组织有明显的毒性损害,结论：连续用肝苏1个月在鸭体内有抗鸭乙型肝炎病毒的作用。     

Liver disease associated with duck hepatitis B virus infection of domestic ducks.

The liver disease associated with duck hepatitis B viremia was investigated in naturally infected ducks from Chi-tung county in China and in both naturally and experimentally infected ducks from the United States. Liver and serum specimens of adult Chinese ducks were examined for duck hepatitis B virus (DHBV) DNA by dot and gel blot hybridization. DHBV was found in serum and (in episomal form only) in livers of 6 of 11 birds exhibiting various degrees of chronic hepatitis. In 1 bird with hepatocellular carcinoma, DHBV DNA was detected at the limit of assay sensitivity and in another not at all, contrasting with findings in humans and woodchucks. In work with California Pekin and Khaki Campbell ducks, known amounts of DHBV were injected into the egg 10 days before, or into ducklings 1 day after, hatching and the livers were examined 6 weeks later. The majority of the injected ducklings had viremia detectable by hybridization 1 or 2 weeks after injection. The presence but not the amount of viremia correlated with incidence and degree of hepatitis, determined under code. The most severe instances of hepatitis, all in Pekin ducks, resembled the hepatitis in adult Chinese ducks of Chi-tung county. Severe and moderate hepatitis were found only in indoor-caged injected animals with viremia and in some uninjected birds without viremia that had been kept in outdoor flocks. The latter hepatitis, as some hepatitis in adult Chinese ducks, may not be related to DHBV. Mild and insignificant hepatitis were also found in injected and noninjected ducklings, some of which had the vertically transmitted spontaneous viremia previously described. The good correlation of experimentally induced viremia with incidence and severity of hepatitis in the Pekin duckling provides a simple, rapid, and relatively inexpensive model to study the relation of lesions to hepatitis B family infection in nonprimates.     

X gene-related sequences in the core gene of duck and heron hepatitis B viruses.

The genomes of all known mammalian hepadnaviruses contain an open reading frame (ORF), designated X, located just upstream from the gene encoding the major viral nucleocapsid polypeptide. This gene is believed to have one or more roles central to the life cycle of these viruses. Consequently, it is surprising that avian hepadnaviruses appear to lack this ORF. However, the observation that the size and position of the core gene in the duck hepatitis B virus (DHBV) genome and the heron hepatitis B virus (HHBV) genome is comparable to the combined X and core genes of the mammalian hepadnaviruses suggests that X function(s) may be performed by the major nucleocapsid polypeptide of DHBV and HHBV. Computer-assisted analyses were carried out to test the hypothesis that the primary and secondary structural characteristics of the X gene product are also present in the major core gene product of the duck (DHBcAg) and heron (HHBcAg) viruses. Primary sequence comparison of the major core-associated polypeptides encoded by the avian and mammalian hepadnaviruses demonstrates considerable homology at both the amino- and carboxyl-terminal regions of these components. However, the middle portion of the DHBcAg and HHBcAg polypeptide, spanning about half the molecule, is unique. Comparison of this region with the carboxyl-terminal half of the X gene sequences from mammalian hepadnaviruses demonstrates similarities in both primary sequence and secondary structural characteristics. These results suggest that X-like gene product sequences are present in the core gene products of DHBV and HHBV. In addition, a sequence of about two dozen residues at the amino terminus of the mammalian X gene product, overlapping the polymerase gene product, is found in the corresponding position in DHBV. This is consistent with the conclusion that the relationship between the DHBV and HHBV core genes compared to the X and core genes of the mammalian hepadnaviruses may be explained by one or more translocations in the this region of the viral genome. The previous finding of X antigen determinants associated with one or more core-related polypeptides in the mammalian hepadnaviruses, combined with the results of this study, suggests that X gene product function is conserved among these viruses.     

Studies on duck hepatitis B virus: Identification and epidemiological survey in Taiwan

The presence of duck hepatitis B virus (DHBV) in domestic ducks in Taiwan was confirmed by DNA polymerase assay, Southern blot analysis and electron microscopy. To investigate the epidemiology of this virus, a total of 1274 serum samples were collected from 30 duck farms from different areas of Taiwan and studied by spot hybridization and/or DNA polymerase assay. The positive rates varied among different strains of ducks: 16% in 243 Pekin ducks, 12% in 392 Chinese common domestic ducks, 4% in 196 Muscovy, 25% in 292 Taiwan Kaiyas and 13% in 151 mule ducks. The positive rate was much higher in the younger ducks; it was highest (30.7%) in ducklings under 1 month of age, followed by ducks aged 1–12 months (11.8%), and lowest in those ducks older than 1 year (7.7%). It was concluded that the prevalence of DHBV infection in domestic ducks in Taiwan is generally high, and that the infected ducks may serve as an animal model for human hepatitis B virus infection which is also prevalent in Taiwan.