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1.
通过构建乙型肝炎病毒(Hepatitis B virus, HBV)单倍体分子,阐明HBV基本核心启动子突变(A1762T/+G1764A,BCP)对HBV复制的影响。本研究收集了HBeAg阴性的慢乙肝患者,将其中两例BCP突变的序列通过重叠PCR克隆到HBV1.3倍体的复制质粒;构建环状的HBV单倍体分子,转染Huh7.0细胞后,通过ELISA、实时荧光定量PCR等分析BCP突变对病毒复制的影响;通过荧光素酶实验分析BCP突变对HBV preC mRNA转录的影响。55例HBeAg阴性的慢乙肝患者中有30例带有A1762T/G1764A的突变;克隆带有BCP突变的临床序列到HBV 1.3倍体转染细胞后,上清中HBeAg为阴性,表面抗原降低10%(P>0.05);构建的HBV单倍体环化后形成闭合环状的cccDNA分子,转染细胞后,结果表明BCP突变后,HBeAg下降55.48%(P<0.05),表面抗原无显著性差异(P>0.05),细胞内病毒颗粒中DNA水平为野生型的3.18倍,上清中HBV DNA为野生型的1.36倍;BCP突变后preC mRNA水平降低81.05... 相似文献
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目的探讨乙型肝炎病毒(HBV)基因型、基本核心启动子(BCP)区双突变(简称BCP双突变)和肝硬化(LC)、肝细胞癌(HCC)发生的关联,分析BCP双突变和HCC临床病理特征的关系。方法随机收集233例慢性HBV感染者的血清,其中80例为慢性乙型肝炎(CHB)患者、75例为LC患者、78例为HCC患者,并系统整理患者的常规检查和病理等资料。采用实时荧光定量聚合酶链反应(FQ-PCR)检测BCP双突,用特异性引物多重PCR扩增确定HBV基因型。用SPSS 11.0分析结果。结果 HBV基因型结果均为B和C型,分别在CHB和LC组,CHB和HCC组间分布差异有统计学意义(P<0.05),在LC和HCC组间分布差异无统计学意义(P>0.05),感染C基因型与LC和HCC发生相关(分别OR=2.73,95%CI=1.29~5.82;OR=2.00,95%CI=0.98~4.09)。BCP双突变也分别在CHB和LC组,CHB和HCC组间分布差异有统计学意义(P<0.05),在LC和HCC组间分布差异无统计学意义(P>0.05),双突变与LC和HCC发生相关(分别OR=1.91,95%CI=0.96~3.82;OR=2.05,95%CI=1.04~4.06)。BCP双突变和伴肝硬化的HCC相关(P<0.05)。结论感染HBV C基因型、BCP双突变可能均是LC和HCC发生的危险因素,BCP双突变可作为LC和HCC早期预警生物标记物。 相似文献
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为研究福建省慢性HBV感染者HBV基因多样性及变异规律,了解该人群HBV-DNA的病毒学特征。收集慢性HBV感染者血清标本,通过巢式PCR法扩增其HBV基因序列,比对NCBI数据库中标准基因型序列,分析HBV基因S区,基本核心启动子区(BCP)及前C区的序列变异情况,并对这些变异可能造成的病毒抗原表达,疫苗逃逸,患者病症改变等情况进行探讨。最终成功扩增82例HBV全长基因序列,其中B基因型56例,C基因型26例。基因组特定功能区序列分析发现慢性HBV感染者HBV基因在S区(23.2%)、BCP区(61.0%)和前C区(29.3%)均出现了不同程度的变异。其中主蛋白(HBsAg)主要抗原决定簇a决定簇45.8%位点出现了变异,这些变异位点中包括与肝炎重症化及免疫逃逸密切相关的位点(aa126、aa129、aa145等)。位点G1896A(19.5%),G1764A(11.0%)和A1762T(9.8%)依然是BCP/前C区的主要突变位点。而位点A1752G(25.6%)高突变率的出现在BCP区应引起关注。此外位点G1764A(χ~2=5.742,P=0.030)、A1896G(χ~2=14.392,P=0.000)以及A1762T/G1764A(χ~2=7.289,P=0.012)的突变更容易发生在HBeAg阴性的样品中;而位点A1846T(χ~2=11.882,P=0.003)、A1762T(χ~2=6.561,P=0.038)和A1896G(χ~2=6.958,P=0.030)的突变与HBV-DNA的病毒载量存在一定相关性。总之,福建省慢性HBV感染者在HBV不同基因功能区域均存在不同程度的变异,一些与HBeAg表达情况、HBV-DNA载量、疫苗免疫逃避及肝细胞癌发生具有相关联的变异位点已经出现,BCP区A1752G位点的高频率出现应值得关注,对于这些变异位点的患者应加强监测。 相似文献
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乙型肝炎病毒的核心启动子各区段功能的研究 总被引:1,自引:0,他引:1
将一系列核心启动子区的缺失突变引入乙肝病毒(HBV)线性转录单元,从病毒的抗原,RNA以及子代DNA等各个水平,分析了各缺失突变对前基因组RNA和前核心RNA转录的影响,对核心启动子各片段的功能进行了深入的研究。C片段缺失后检测不到e抗原和前核心RNA,却仍有核心抗原和前基因组RNA的合成以及病毒子代DNA的复制;而B3片段缺失后e抗原和核心抗原均有显著下降,但仍能检测到两种mRNA的合成和病毒子 相似文献
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目的建立检测肺炎支原体(Mycoplasma pneumoniae,MP)23S rRNA V区2063基因型的反向斑点杂交方法,并与PCR产物直接测序法的结果进行比较分析。方法19例自临床咽拭子标本分离培养的MP,用自行设计的反向斑点杂交法检测23S rRNA V区2063基因型,同时对相应序列测序分析。抽提标准菌株FH和127份经PCR测定MP阳性的临床标本基因组DNA,用MP阴性的基因组DNA抽提物5份作阴性对照,用反向斑点杂交法检测23S rRNA V区2063基因型。结果19例分离培养的MP,经反向斑点杂交法检测15株有23S rRNA V区基因A2063G位点突变;4株为2063A,与测序结果一致(Kappa一致性检验,P〉0.05)。经反向斑点杂交法检测,127份MP阳性的基因组DNA抽提物中有122份标本为A2063G位点突变,标准菌株FH和2份DNA抽提物的2063位点碱基为A,还有3份抽提物标本的2063位点碱基既有A又有G,5份阴性对照均无显色。结论反向斑点杂交方法能快速、准确检测临床MP 23S rRNA V区2063基因型。 相似文献
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肝癌组织中HBV核心基因启动子突变的研究 总被引:2,自引:0,他引:2
为深入了解乙肝病毒(HBV)致癌机理,用套式PCR对广西14例血清HBV DNA阳性的原发性肝癌患者癌组织、10例乙型病毒性肝炎患者血清及10例乙肝病毒无症状携带者血清HBV核心基因启动子进行扩增,阳性者用Sanger氏双脱氧法做序列分析,发现肝癌组织10例PCR阳性,阳性率71.4%,所有PCR阳性标本的整合体均有乙肝病毒核心基因启动子双突变序列(nt 1 762 A→T, 1 764 G→A),并且在其周围各序列都有不同部位的点突变,标本C14核苷酸的缺失突变高达10个.乙肝患者6例PCR阳性,其中3例乙肝病毒核心基因启动子发生双突变.无症状携带者中4例PCR阳性,其中仅1例发生双突变.结果提示乙肝病毒核心基因启动子双突变在肝癌患者中较常见,肝炎患者次之,无症状携带者居最后. 相似文献
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为深入了解乙肝病毒 (HBV)致癌机理 ,用套式PCR对广西 14例血清HBVDNA阳性的原发性肝癌患者癌组织、10例乙型病毒性肝炎患者血清及 10例乙肝病毒无症状携带者血清HBV核心基因启动子进行扩增 ,阳性者用Sanger氏双脱氧法做序列分析 ,发现肝癌组织 10例PCR阳性 ,阳性率 71.4 % ,所有PCR阳性标本的整合体均有乙肝病毒核心基因启动子双突变序列 (nt 176 2A→T ,176 4G→A) ,并且在其周围各序列都有不同部位的点突变 ,标本C14核苷酸的缺失突变高达10个。乙肝患者 6例PCR阳性 ,其中 3例乙肝病毒核心基因启动子发生双突变。无症状携带者中 4例PCR阳性 ,其中仅 1例发生双突变。结果提示乙肝病毒核心基因启动子双突变在肝癌患者中较常见 ,肝炎患者次之 ,无症状携带者居最后。 相似文献
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反向斑点杂交法快速检测HBV基因型反应条件的优化和建立* 总被引:1,自引:0,他引:1
利用反向斑点杂交技术,设计出特异性基因分型探针,并将探针固定在带正电荷的尼龙膜上,与PCR扩增带有地高辛标记的临床血清样本进行杂交。通过优化杂交反应条件,建立起简单、快速、特异地检测HBV基因型的方法。利用该方法对重庆地区临床样本进行分型检测,并与直接测序结果比较。结果表明新建的HBV基因分型方法可对拷贝数在103以上的血清样本准确分型,特异性达到96.67%。重庆地区感染HBV主要以B型为主。 相似文献
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为深入了解乙肝病毒(HBV)致癌机理,用磁式PCR对广西14例血清HBV DNA阳性的原发性肝癌患者癌组织、10例乙型病毒性肝炎患者血清及10例乙肝病毒无症状携带者血清HBV核心基因启动子进行扩增,阳性者用Sanger氏双脱氧法做序列分析,发现肝癌组织10例PCR阳性,阳性率71.4%,所有PCR阳性标本的整合体均有乙肝病毒核心基因启动子双突变序列(nt1762A→T,1764G→A),并且在其财 相似文献
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Dong Q Chan HL Liu Z Chan DP Zhang B Chen Y Kung HF Sung JJ He ML 《Biochemical and biophysical research communications》2008,374(4):773-776
Hepatitis B virus (HBV) infection is a major global health problem that causes over one million deaths annually. A1762T and G1764A mutations in the basal core promoter are often present in HBV patients but seldom in asymptomatic carriers, and are highly correlated with the increased risk of HBV-associated hepatocellular carcinoma (HCC). In this study, for the first time, we show that the basal core promoter activity of HBV strains isolated from asymptomatic carriers is decreased when 1762A is mutated to 1762T or 1764G is mutated to 1764A by site directed mutagenesis. By contrast, the promoter activity of HBV strains isolated from HCC patients is increased when 1762T and 1764A are reversely mutated into 1762A and 1764G, respectively. 1764G contributes more promoter activity than 1762T. We also show that T1762A and G1764A double mutations synergize the reduction of the promoter activity. A mechanism of HBV evasion from host immunoresponse that may facilitate disease development is also discussed. 相似文献
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Reverse dot blot hybridization: A useful method for the direct identification of lactic acid bacteria in fermented food 总被引:2,自引:0,他引:2
Abstract A rapid method for a reliable and simultaneous identification of different lactic acid bacteria in fermented food has been developed. Various 16S and 23S rRNA-targeted, species-specific oligonucleotides were applied as capture probes in a non-radioactive reverse dot blot hybridization. A simple and fast DNA extraction method in combination with in vitro amplification of rRNA gene fragments enables the direct detection of typical starter organisms without any preceding enrichment or cultivation steps. Various lactic acid bacteria occurring in cheese, yogurt, sausages, sauerkraut and sourdough could be identified at the species level within 1 day. 相似文献
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Tsai SL Chen PJ Yang PM Liao TH Sung JL Lai MY Huang JH Chang TH Chen DS 《Journal of biomedical science》1994,1(2):105-118
T cell clones specific for hepatitis B core (HBcAg) and e (HBeAg) antigens of hepatitis B virus (HBV) were generated from liver infiltrates of HBeAg-positive patients. Analyzed with a panel of overlapping synthetic peptides spanning the complete sequences of HBcAg and HBeAg, eight clones responded specifically to the e2 peptide (PAYRPPNAPIL; amino acid residues 130–140 of HBcAg and HBeAg), which was doubly restricted by class I and II molecules. A preferential usage of the T cell receptor (TCR) chain variable (V) gene was found: V12.1 for five HLA-Cw9(3)-restricted cytotoxic T lymphocyte (CTL) clones, and V7.1 for three other HLA-DRw52-restricted type 1 helper T cell (Th1) clones. Although heterogeneous in the usage of TCR chain joining region (J) segments, their junctional-region sequences revealed conserved hydrophilic serine residues in seven of the eight e2-specific T cell clones. Single alanine substitution of the centrally located and the only hydrophilic asparagine residue of e2 peptide abrogated T cell responsiveness. The nonstimulatory e2 analogue could competitively inhibit e2-specific responses. These results demonstrate that both CTL and Th1 clones recognizing a determinant of HBcAg and HBeAg are present in the liver of chronic hepatitis B patients. The preferential V gene usage and the expression of conserved residues in junctional-region sequences of TCR chains by viral-peptide-specific, intrahepatic T cells may provide a T cell mechanism of HBV immunopathogenesis. 相似文献
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Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex 总被引:16,自引:0,他引:16
Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus. 相似文献
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Hepatitis B is a potentially life-threatening liver infection caused by the hepatitis B virus (HBV) and is a major global health problem. HBV is the most common serious viral infection and a leading cause of death in mainland China. Around 130 million people in China are carriers of HBV, almost a third of the people infected with HBV worldwide and about 10% of the general population in the country; among them 30 million are chronically infected. Every year, 300,000 people die from HBV-related diseases in China, accounting for 40-50% of HBV-related deaths worldwide. Despite an effective vaccination program for newborn babies since the 1990s, which has reduced chronic HBV infection in children, the incidence of hepatitis B is still increasing in China. We propose a mathematical model to understand the transmission dynamics and prevalence of HBV infection in China. Based on the data reported by the Ministry of Health of China, the model provides an approximate estimate of the basic reproduction number R0=2.406. This indicates that hepatitis B is endemic in China and is approaching its equilibrium with the current immunization program and control measures. Although China made a great progress in increasing coverage among infants with hepatitis B vaccine, it has a long and hard battle to fight in order to significantly reduce the incidence and eventually eradicate the virus. 相似文献
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