中国大陆新发现丙型肝炎病毒3′非编码区终止密码子变异

目的获得全长中国大陆1b型丙型肝炎病毒(HCV)3′非编码区(3′UTR)cDNA,并分析一级结构的变异,为进一步研究其在HCV复制、翻译中的调控机制和开发新的抗HCV药物奠定基础。方法利用逆转录套式聚合酶链反应(RT-PCR)限制性内切酶长度多态性分析(RFLP)初步筛选出1例1b型HCV感染者,采用半套式RT-PCR法扩增出约400bp的cDNA片段,克隆测序。结果获得的全长1b型HCV3′端序列,由高变区、Poly(u)区、Poly(u/c)区及98碱基区4部分组成;首次发现终止密码子突变由TGA突变为TGG,可导致NS5B的翻译不能及时终止。结论半套式RT-PCR法可有效获得病毒基因组的全长末端序列;首次报道终止密码子突变导致NS5B区延长,3′UTR缩短。该发现对了解HCV的复制和翻译机制可能有一定的理论和实践意义。     

中国大陆1b型丙型肝炎病毒3''非编码区序列变异研究

丙型肝炎病毒(HCV)是一类有包膜的单股正链RNA病毒,其基因组全长约9600bp,5’端和3’端各有一非编码区,3’非编码区 (3’UTR)长约220-272bp[1]。自1989年HCV成功克隆以来,已有近百株报告,然而绝大多数是3’末端残缺的克隆,可能已丧失感染能力[2]。我     

丙型肝炎5''''非编码区末端快速基因扩增方法的建立及应用

目的 建立快速获得丙型肝炎（HCV）基因组5’非编码区（5’uTR）真末端序列的分子生物学方法。方法 逆转录后利用末端聚合酶（TOT）进行加尾反应，再利用套式聚合酶链反应（PCR）扩增出目的末端基因的cDNA片段，A-T克隆，用限制性内切酶片段长度多态性分析（RVLP）与PCR鉴定重组子，全自动序列分析仪测定插入子序列。结果 cDNA末端快速扩增技术（RACE）获得5株HCV5’UTR克隆，包括3株全长克隆和2株缺失克隆。2株缺失克隆，一条在5’末端缺失53个碱基，另一条缺失144个碱基。结论 RACE技术快速、有效、实用，可有效获得丙型肝炎病毒基因组的5’非编码区末端序列。     

丙型肝炎病毒5′端非编码区和NS3蛋白酶共调控外分泌性碱性磷酸酶表达细胞模型的建立及意义

目的构建丙型肝炎病毒5′端非编码区(HCV 5′NCR)和NS3丝氨酸蛋白酶共调控外分泌性碱性磷酸酶(SEAP)表达细胞模型,并分析其用于抗HCV药物筛选和评价的可行性.方法用聚合酶链反应技术扩增HCV 5′NCR和NS3/4A片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建含HCV 5′NCR-NS3/4A-SEAP嵌合基因的重组表达质粒pNCR-NS3/4A-SEAP.将重组质粒转染至肝细胞株QSG7701,用化学发光法检测SEAP的表达,并观察HCV 5′NCR区对应的反义寡聚核苷酸(ASODN)和丝氨酸蛋白酶抑制剂TPCK对SEAP表达的影响.结果重组质粒pNCR-NS3/4A-SEAP有高强度SEAP表达,5 μmol/L、10 μmol/L ASODN和100μmol/L TPCK对SEAP表达有显著抑制作用(f值分别为4.315、6.985、6.949,P值均＜0.01).结论重组质粒pNCR-NS3/4A-SEAP的SEAP表达受HCV 5′NCR和NS 3蛋白酶共调控,建立的细胞模型可用于以H CV 5′NCR和NS 3蛋白酶为靶位点的药物筛选和评价.     

丙型肝炎病毒5''端非编码区的5''端序列对其翻译启动活性的影响

目的分析丙型肝炎病毒(HCV)5’端非编码区(5’NCR)的5’端序列对其翻译启动活性的影响。方法用PCR技术扩增获得全长和5’端17个碱基缺失的HCV5’NCR片段,定向克隆至表达质粒pSEAP2Control的SEAP基因上游,分别构建成全长和5’端截短的HCV5’NCR调控SEAP表达的重组质粒pNCRSEAP和pdNCRSEAP。用脂质体方法将重组质粒转染至肝细胞株QSG7701,并用化学发光法检测SEAP的表达。结果酶切和测序结果表明,重组质粒pNCRSEAP和pdNCRSEAP构建成功。表达的发光强度分别为390482±42856和635265±52285RLU,二者有差异显著性(P<0.01)。结论HCV5’NCR的5’端碱基缺失提高了它对SEAP基因的翻译启动活性,为进一步分析HCVIRES的结构提供了实验基础。     

丙型肝炎病毒NS3蛋白人源基因工程单链抗体的表达

目的在大肠杆菌XL1-Blue中表达可溶性的抗HCV非结构蛋白NS3的人源单链可变区抗体（single-chainvariablefragmentantibody,ScFv）。方法以重组的HCVNS3为抗原,利用噬菌体抗体库技术筛选含有抗-HCVNS3ScFv基因的噬菌体克隆。从噬菌体抗体阳性克隆中提取质粒,经SfiI/NotⅠ酶切鉴定后,亚克隆到pCANTAB5E载体;转化大肠杆菌XL1-Blue,提取质粒进行DNA序列测定;异丙基硫代-β-D-半乳糖苷（isopropylthio-β-D-galactoside,IPTG）诱导表达HCVNS3可溶性单链可变区抗体。ELISA和斑点吸印杂交检测其与不同来源的抗原的结合活性。结果筛选到的HCVNS3的单链抗体基因,经限制性内切酶酶切和序列分析表明,该抗体基因由750bp组成,ELISA和斑点吸印杂交结果表明,在大肠杆菌XL1-Blue中表达的HCVNS3的单链抗体,可与不同来源的NS3抗原结合。结论大肠杆菌XL1-Blue表达的NS3-ScFv具有结合不同来源的HCVNS3的活性和特异性。     

丙型肝炎病毒5''非翻译区DNA序列在HepG2细胞中的启动子活性

目的研究丙型肝炎病毒(HCV)基因组5'非翻译区(5'UTR)DNA序列在HepG2细胞中的启动子活性,以了解HCV的复制调控机制.方法分别构建HCV基因组5'UTR DNA正反向序列驱动虫荧光素酶基因表达的质粒5'UTR- Luc(+)/(-)和5'UTR DNA序列驱动绿色荧光蛋白基因表达的质粒5'UTR -EGFP(+)/(-),分别转染HepG2细胞,用双荧光素酶检测系统检测虫荧光素酶的表达水平,逆转录聚合酶链反应检测虫荧光素酶基因m R N A水平,荧光显微镜观察绿色荧光蛋白基因的表达水平,并与相应对照作比较,来证实H CV基因组5'UTR DNA序列的启动子活性.结果 5'UTR- LUc(+)有明显的虫荧光素酶表达,但比pGL3 control表达水平低(Luc/R为0.690 ± 0.086,Luc/RL为4.210±0.340),而5'UTR-Luc(-)和pGL3 enhancer无明显虫荧光素酶表达(Luc/RL分别为0.095±0.008和0.044±0.00 5);逆转录聚合酶链反应结果与之相符,5'UTR- Luc(+)检测到虫荧光素酶基因mRNA,而5'UTRLuc(-)则未检测到.5'UTR- EGFP(+)观察到较强绿色荧光,而5'UTR -EGFP(-)无荧光表达.结论 HCV基因组5'UTR DNA序列具有明显的启动子活性,能启动下游基因的表达,在HCV基因组复制过程中有重要作用.     

中国兰州地区丙型肝炎病毒感染的基因型分布特征

目的了解中国兰州地区丙型肝炎病毒（HCV）感染基因型分布特点。方法筛选41例来自兰州的HCVRNA阳性患者血清样品,扩增HCV5′非编码区（5’NCR）,得到257bp的聚合酶链反应（PCR）产物,并对这些产物进行测序。然后将所得序列与GenBank参考序列比对,并作同源性分析和进化分析。结果 41例样品中共检出1b型感染25例（61.0%）,1c型感染8例（19.5%）,2a型4例（9.8%）,1a型3例（7.3%）,3b型感染1例（2.4%）。结论兰州地区的基因型分布以1b型为主,并首次在中国检出1c型感染,且感染率仅低于1b型;此外还检出2a、1a和3b;未检出4、5和6型等基因型。     

丙型肝炎病毒NS3区基因的克隆、表达及产物的纯化和抗原性分析

目前，我国阻断丙型肝炎传播的主要方法是用抗丙型肝炎病毒(HCV)酶联免疫吸附法(ELISA)试剂筛选供血员，国产试剂盒所用NS3抗原质量不太理想，使得国产试剂对NS3抗体漏检较多，提高该试剂盒质量的当务之急是开发更好的14CV抗原，特别是非结构区抗原。为分析和比较不同长度和型别的NS3片段的抗原性，我们在大肠杆菌中表达了5个不同长度和型别的HCV NS3抗原，并作为包被抗原，按间接ELISA原理检测抗HCV国家参考品血清及部分临床样本，比较其抗原性。     

丙型肝炎病毒5′非翻译区 DNA 序列在 HepG2细胞中的启动子活性

目的 研究丙型肝炎病毒(HCV)基因组5′非翻译区(5′UTR)DNA 序列在 HepG2细胞中 的启动子活性，以了解 HCV 的复制调控机制。方法 分别构建 HCV 基因组5′UTR DNA 正反向序列驱动 虫荧光素酶基因表达的质粒5′UTR-Luc(+)/(-)和5′UTR DNA 序列驱动绿色荧光蛋白基因表达的质粒5′ UTR-EGFP(+)/(-)，分别转染 HepG2细胞，用双荧光素酶检测系统检测虫荧光素酶的表达水平，逆转录 聚合酶链反应检测虫荧光素酶基因 m R N A 水平，荧光显微镜观察绿色荧光蛋白基因的表达水平，并与相应 对照作比较，来证实 HCV 基因组5′UTR DNA 序列的启动子活性。结果 5′UTR-Luc(+)有明显的虫 荧光素酶表达，但比 pGL3 control 表达水平低(Luc/R为0．690±0．086，Luc/RL 为4．210±0．340)，而5 ′UTR-Luc(-)和 pGL3 enhancer 无明显虫荧光素酶表达(Luc/RL 分别为0．095±0．008和0．044±0． 005)；逆转录聚合酶链反应结果与之相符，5′UTR-Luc(+)检测到虫荧光素酶基因 mRNA，而5′UTR- Luc(-)则未检测到。5′UTR-EGFP(+)观察到较强绿色荧光，而5′UTR-EGFP(-)无荧光表达。 结论 HCV 基因组5′U TR DNA 序列具有明显的启动子活性，能启动下游基因的表达，在 HCV 基因组复 制过程中有重要作用。     

A novel stop codon mutation in HBsAg gene identified in a hepatitis B virus strain associated with cryptogenic cirrhosis

AIM: HBsAg is the most important serological marker for acute or chronic hepatitis B. Nevertheless, there were reports of HBsAg-negative infection caused by hepatitis B virus in recent years. We had a patient with crytogenic cirrhosis who was negative for HBsAg, positive for anti-HBs and HBeAg.This paper was to explore the pathogenic and molecular basis of the unusual serological pattern.METHODS: HBV serologic markers were qualitatively and quantitatively determined. HBV DNA in serum was qualitatively tested using routine Polymerase chain reaction (PCR), and the viral level was determined with real-time fluorescence quantitative PCR. HBsAg gene was amplified and cloned. Four clones were sequenced. The new genomic sequences were compared with GenBank on the DNA level as well as the protein level.RESULTS: The qualitative results of serological markers were HBsAg(-), anti-HBs(+), HBeAg(+), anti-HBe(-) and anti-HBc(+). The quantitative results of serological marker were HBsAg (S/N): 0.77 (cut off of S/N: ≥2.00), HBeAg (S/N): 56.43 (cut off S/N: ≥2.10), anti-HBc (S/Co): 2.03 (cut off of S/Co: ≤ 1.00). The viral level was as high as 1.54×109copies/ml. Sequencing of the HBsAg gene clones revealed a unique point mutation at nucleotide 336 (C to A), which resulted in a novel stop codon at aa 61. The novel HBsAg gene stop mutation had not been described.CONCLUSION: The lack of detection of HBsAg in the presence of high viral levels of replication may be caused by the existence of viral genomes harboring point mutations which resulted in stop codon upstream of the″a″ determinant in HBsAg gene.     

包含YMDD基因序列的乙型肝炎病毒P区基因变异的研究

目的研究包含YMDD基因序列的乙型肝炎病毒(HBV)P区基因的一级结构及其变异特点。方法从4例末经任何抗病毒治疗的HBV感染患者外周血血清中扩增HBV P基因区序列(长度为1057bp)，双酶切后克隆入pUC19载体中，每份标本随机取20个经限制性片段长度多态性分析(RFLP)法鉴定为阳性的克隆，采取错配聚合酶链反应限制性片段长度多态性分析(PCR-RFLP)法检测YMDD基序变异，最后取其中两个标本共10个阳性克隆进行双向测序。结果标本1和2克隆间核苷酸变异率分别为0．3％～1．1％，0．4％～1．7％。80个阳性克隆中，有2个克隆YMDD基序变异为YMGD。结论包含YMDD基序的HBVP区存在准种分布特征，在未经抗病毒治疗的患者血清中，存在一种新的YMDD变异形式，即YMGD变异。     

一种新的乙型肝炎病毒X基因变异方式

目的:证实乙型肝炎病毒(hepatitis B virus,HBV)X基因一种新的变异方式.方法:从HBV慢性感染患者血清中提取HBVDNA,扩增X基因序列,克隆入pMD19 T载体,选择阳性克隆进行DNA测序,与已知HBV基因相应序列比较该患者体内HBV基因变异位点以及变异形式.结果:从21例患者中共挑选74个克隆测序,测序结果提示54个克隆X基因下游大段缺失突变,长度达234 nt,位于1601-1834 nt处,另有1个克隆发生245 nt缺失突变.发生缺失变异的病毒株同时存在G/A1515C、G1518C和A1585T替换突变,这两种突变具有联动特征.缺失突变株HBx仅编码76 aa其第44和45位编码为LL,具有特异性.结论:观察到一种X蛋白变异方式,这种大段缺失突变导致X蛋白下游编码序列丢失,其为X因子还是X蛋白以及这种变异是否为常态形式尚需进一步研究.     

Effect of mutation in the hepatitis C virus nonstructural 5B region on HCV replication

Background We have reported that the presence of a mutation at the hepatitis C virus (HCV) nonstructural protein 5B (NS5B), defined as a change in amino acids at sites specific for a different reported genotype, was related to complete response (CR) to interferon (IFN) therapy in patients with chronic hepatitis C (CHC) with genotype 1b. The present study assessed the impact of the NS5B mutation on the replication of HCV in these patients.Methods Genotype-specific mutations of HCV NS5B were determined by direct sequencing. We measured HCV-RNA titers in serum by real-time detected polymerase chain reaction (PCR), and serum HCV core protein levels (as a marker of HCV-RNA replication) were measured using an enzyme immunoassay in patients with CHC genotype 1b. RNA-dependent RNA polymerase (RdRp) activity was measured by Behrens method in liver cirrhosis patients infected with HCV (n = 13) and in those infected with hepatitis B virus (HBV; n = 2).Results The titers of HCV-RNA (n = 44) and the levels of HCV core protein (n = 41) were significantly lower in patients with the HCV genotype 1b mutant compared with wild-type HCV (P < 0.05). RdRp activity in liver tissue did not show any correlation with the HCV NS5B mutation.Conclusions HCV NS5B genotype-specific mutations in HCV genotype 1b may influence HCV replication.     

Site-specific mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication

The number of amino acid substitutions in the interferon sensitivity-determining region (ISDR) in the nonstructural 5A (NS5A) gene of hepatitis C virus (HCV) is closely associated with the interferon (IFN) response and viral load. Several HCV replicon-based studies have reported that ISDR sequences had an influence on viral replication in vitro. However, it is unclear as to how different ISDR sequences affect HCV replication. Various clinically observed ISDR sequences were introduced into HCV replicons and their contribution to viral replication was investigated using a colony formation assay and/or a transient replication assay. A mapping study of the ISDR was performed to identify the amino acid positions that critically affect replication. While no colonies were formed in the colony formation assay using HCV replicons with few mutations (0, 1 and 3) in the ISDR, numerous colonies (>200) appeared when using constructs with six mutations. Introduction of various distinct ISDR sequences with multiple mutations resulted in replication enhancement in transient assays. A mapping study identified several specific sites in the ISDR that critically affected replication, including codon 2209 which, in patients, was closely associated with a strong response to IFN. ISDR sequences associated with a clinical IFN response and viral load modulated the replication of HCV replicons, suggesting the importance of the ISDR sequence in HCV infection.     

一种新的HBeAg阴性慢性乙型肝炎病毒变异机制

Objective To investigate mutation patterns in core promoter(CP)region of hepatitis B virus(HBV).Methods HBV DNA was extracted from sera of patients with chronic HBV infection.The CP sequence was amplified by polymerase chain reaction(PCR)and cloned into pMD19 T vector.The positive clones were then sequenced.The sequences were compared with known HBV genome in GenBank to identify the mutation sites and patterns of patients with chronic HBV infection.Results There were 74 clones from 21 patients with chronic HBV infection which were sequenced.The sequence comparisons showed that there was a 234-nucleotide deletion in CP region of HBV genome in 54 clones and a 245-nucleotide deletion in one clone.These deletion regions included CP,HBeAg initiation codon and direct repeat sequence(DR)Ⅰ regions,which named CP deletion(CPD).A1585T replacement mutation was also found in HBV strain with CPD,which indicated that there was linkage between these two mutations.Conclusions A novel mechanism of HBeAg negative chronic hepatitis B is observed,which includes deletions of CP and HBeAg initiation codon.Meanwhile,a simple and useful PCR method is developed to detect CPD.     

一种新的HBeAg阴性慢性乙型肝炎病毒变异机制

Objective To investigate mutation patterns in core promoter(CP)region of hepatitis B virus(HBV).Methods HBV DNA was extracted from sera of patients with chronic HBV infection.The CP sequence was amplified by polymerase chain reaction(PCR)and cloned into pMD19 T vector.The positive clones were then sequenced.The sequences were compared with known HBV genome in GenBank to identify the mutation sites and patterns of patients with chronic HBV infection.Results There were 74 clones from 21 patients with chronic HBV infection which were sequenced.The sequence comparisons showed that there was a 234-nucleotide deletion in CP region of HBV genome in 54 clones and a 245-nucleotide deletion in one clone.These deletion regions included CP,HBeAg initiation codon and direct repeat sequence(DR)Ⅰ regions,which named CP deletion(CPD).A1585T replacement mutation was also found in HBV strain with CPD,which indicated that there was linkage between these two mutations.Conclusions A novel mechanism of HBeAg negative chronic hepatitis B is observed,which includes deletions of CP and HBeAg initiation codon.Meanwhile,a simple and useful PCR method is developed to detect CPD.