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。     

抗人精浆蛋白单链抗体基因的构建及序列分析

目的：构建抗人精浆蛋白的单链抗体基因，可为进一步构建、表达抗人精浆蛋白单链抗体／羧肽酶Ａ融合蛋白，用于前列腺癌的抗体导向酶－前体药物疗法奠定基础。方法：利用加端ＰＣＲ技术，在已克隆的抗人精浆蛋白单克隆抗体ＶＨ和Ｖκ基因两端加上限制性酶切位点，再分别克隆入载体ｐＵＣ１９－ｌｉｎｋｅｒ中，构建ＶＨ－ｌｉｎｋｅｒ－Ｖκ形式的Ｅ４Ｂ７单链抗体基因。利用全自动荧光测序仪测定其序列，采用ＰＣ／Ｇｅｎｅ软件与已知的ＶＨ和Ｖκ基因进行核苷酸序列的同源性比较，并推导其编码的氨基酸序列。结果：Ｅ４Ｂ７单链抗体基因全长为７４１ｂｐ，为一开放读框，编码２４７个氨基酸，与已知的抗人精浆蛋白单克隆抗体ＶＨ和Ｖκ基因完全同源。ＶＨ和Ｖκ间有４５ｂｐ的ｌｉｎｋｅｒ序列，推导的氨基酸序列为（Ｇｌｙ４Ｓｅｒ）３，与设计的序列相符。结论：构建成功序列正确的抗人精浆蛋白单链抗体基因，为构建、表达抗人精浆蛋白／羧肽酶Ａ双功能抗体奠定了基础     

散发性戊型肝炎病毒部分核苷酸序列分析

目的为阐明戊型肝炎病毒（ＨＥＶ）毒株的地理分布、流行特征以及研制血清学诊断试剂和基因工程疫苗提供资料。方法选择１例浙江仙居山区散发性ＨＥ患者，从其血清中分离ＨＥＶＲＮＡ，通过逆转录－套式聚合酶链反应法（ＲＴ－ｎＰＣＲ），扩增该散发性ＨＥＶ（Ｚ－３３株）ＯＲＦ２区部分ｃＤＮＡ片段（４９７ｂｐ），然后进行直接序列分析，并与ＨＥＶ各主要代表株序列作比较。结果Ｚ－３３株与新疆流行株ＣＨ１．１的核苷酸及氨基酸序列同源性分别为９６．７％及９８．２％；与缅甸流行株的同源性分别为９４．２％及９９．１％；与缅甸散发株的同源性分别为９５．２％及９９．１％，与墨西哥株的同源性分别为８１．８％及９４．５％。结论浙江仙居散发性ＨＥＶＺ－３３株和新疆流行株ＣＨ１．１一样，与ＨＥＶ缅甸株可能为同一亚型。     

丙型肝炎病毒1b型NS5 A区基因结构变异与α干扰？…

目的 观察丙型肝炎患者丙型肝炎病毒（ＨＣＶ）１ｂ型基因组部分ＮＳ５ Ａ区核苷酸,氨基酸的变异情况并探讨其与α干扰素疗效的相关性。方法 患者干扰素治疗前,中,后留血标本,用聚合酶链反应（ＰＣＲ）扩增ＨＣＶ病毒ＮＳ５ Ａ区部分基因片段并用直接测序法测序。与ＨＣＶ－Ｊ株及ＨＣＶ－河北株（ＨＣＶ－ＨＢ）比较核苷酸及氨基酸序列的同源性,根据α干扰素疗效分析ＨＣＶ１ｂ是否存在干扰素敏感决定区。     

猪型流感病毒血凝素基因的核苷酸全序列分析

对我国大陆首次从猪群中分离到的猪型（Ｈ１Ｎ１）流感病毒血凝素（ＨＡ）基因核苷酸全序列进行了测定，其长度为１７７８ｂｐ，共编码５６６个氨基酸，其中信号区１７个，ＨＡ１区３２６个，多肽连接区１个，ＨＡ２区２２２个。与Ａ／ＮＪ／１１／７６（Ｈ１Ｎ１）毒株ＨＡ蛋白分子上氨基酸序列相比，其同源性多１个糖基化点，然而其余的包括２个重叠的糖基化点均相同。     

庚型肝炎病毒(HGV)E2区cDNA的克隆与表达

目的　克隆ＨＧＶ Ｅ２ 基因并在原核细胞系统中表达ＨＧＶ Ｅ２ 蛋白。方法　从ＨＧＶＲＮＡ 阳性血浆中抽提病毒ＲＮＡ,利用半巢式ＲＴＰＣＲ 法扩增ＨＧＶ Ｅ２ 基因片段并进行序列分析。然后将该片段克隆到ｐＧＥＸ５ｘ１ 表达载体上,进行诱导表达。表达产物用抗ＨＧＶ 阳性血浆作Ｗｅｓｔｅｒｎ ｂｌｏｔ 活性鉴定。结果　获得ＨＧＶ Ｅ２ Ｎ 端长度为７７９ 个核苷酸的基因片段。该片段与美国株ＨＧＶ（Ｕ４４４０２） 、西非株ＧＢＶＣ（ Ｕ３６３８０） 、中国株ＨＧＶ（ Ｕ７５３５６） 的核苷酸序列同源性分别为８４ ％ 、８５ ％ 、９１ ％ ,推测的氨基酸序列同源性分别为８８ ％ 、９３ ％ 、９４ ％ 。表达产物为相对分子质量约５０ ×１０３的ＧＳＴＥ２ 融合蛋白,在细胞内形成包涵体。Ｗｅｓｔｅｒｎ ｂｌｏｔ 反应中在约５０ ×１０３ 处有显色条带。结论本研究成功地在原核细胞系统中表达了具有抗原性的ＨＧＶ Ｅ２ 蛋白,为进一步研究ＨＧＶ Ｅ２ 蛋白及Ｅ２ 抗体的生物学功能打下了基础。     

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。     

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聚合酶链反应从１例中国庚型肝炎患者血清中扩增出仿前区部分基因及Ｅ１区５‘端共３０９ｂｐ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。     

中国狂犬病固定毒aG株N,NS和M蛋白基因的核苷酸序列测定

报告了中国狂犬病固定毒ａＧ株核衣壳蛋白（Ｎ蛋白），核蛋白壳磷酸蛋白（ＮＳ蛋白，又称Ｍ１蛋白）和基质蛋白（Ｍ蛋白，又称Ｍ２蛋白）蛋白基因的核苷酸序列和氨基酸推导序列。各基因编码区的全长分别是：Ｎ基因１３５２，ＮＳ基因８９４和Ｍ基因６０９个核苷酸。ａＧ和ＣＶＳ株的Ｎ，ＮＳ和Ｍ基因的核苷酸序列都具有高的同源性，分别为９１．９５％，９０．２７％和９０．８０％。     

中国狂犬病固定毒aG株N,NS和M蛋白基因的核苷酸序…

报告了中国狂犬病固定毒ａＧ株核衣壳蛋白（Ｎ蛋白），核蛋白壳磷酸蛋白（ＮＳ蛋白，又称Ｍ１蛋白）和基质蛋白（Ｍ蛋白，又称Ｍ２蛋白）蛋白基因的核苷酸序列和氨基酸推导序列。各基因编码区的全长分别是：Ｎ基因１３５２，ＮＳ基因８９４和Ｍ基因６０９个核苷酸。ａＧ和ＣＶＳ株的Ｎ，ＮＳ的Ｍ基因的核苷酸序列都具有高的同源性，别为９１．９５％，９０．２７％和９０．８０％。     

丙型肝炎病毒包膜糖蛋白高变区1多抗原肽设计及？…

目的 应用多抗原肽（ＭＡＰ）研究丙型肝炎病毒包膜糖蛋白高变区１（ＨＶＲ１）的抗原性。方法 根据已经获得的ＨＣＶ－ＢＪ株Ｅ２／ＮＳ１区氨基酸序列,参照国内外得所报道的ＨＶ ＨＶＲ１序列及抗原性参数,设计并合成含ＨＣＶ ＨＶＲ１３９０－４１１ａａ序列２２个氨基酸的线性表位多肽（以ＬＰ表示）及ＭＡＰ（对称８分枝）,分别以ＬＰ和ＭＡＰ免疫Ｂａｌｂ／Ｃ小鼠及家兔,比较其免疫原性。结果 ＭＡＰ免疫原性明显强于     

Predominance of antibodies to hepatitis C virus envelope proteins in various disease statuses of hepatitis C

The antibody profile to various proteins of hepatitis C virus (HCV) was studied in 113 patients positive for HCV RNA in various disease statuses of hepatitis C (HC). A single peptide (E2/NS1, aa 413-436 of HCV polyprotein) chosen from a conserved region at the C-terminus of the hypervariable region (HVR) HVR1 of HCV was found to be sufficient for reliable diagnosis of the infection, even in the acute phase. Six hundred and one suspected HC cases and 200 voluntary blood donors were tested by this peptide. The sensitivity of detection of HCV antibodies by this peptide did not increase with addition of peptides from other HCV proteins. Our results clearly demonstrate that antibodies to HCV envelope proteins occur in a higher percentage of the infected population than those to other proteins. This emphasizes the necessity of using representative sequences from HCV envelope proteins in diagnostic immunoassays of this viral infection.     

Analysis of mother-to-infant transmission of hepatitis C virus: Quasispecies nature and buoyant densities of maternal virus populations

Mother-to-infant transmission of hepatitis C virus (HCV) was analyzed by sequencing of viral RNA and semiquantitative polymerase chain reaction following ultracentrifugation of maternal sera. In two mother-infant pairs, the hypervariable region 1 (HVR1) and carboxyl terminus of envelope 1 (E1) were sequenced. Both viral sequences in the infants were less diverse than those of their mothers. Although the E1 sequences were almost identical in each mother-infant pair, the HVR1 sequences of the infants were related, but not identical, to those of the mothers. Serial examinations of one infant revealed that the HVR1 nucleotide sequence did not change from 10 days to 3 months of age. In six mothers with uninfected infants, all of the dense fractions of sera contained significant amounts of HCV RNA, whereas in six mothers with infected infants, only two of those fractions contained significant amounts of HCV RNA. These results indicate that the strains of HCV detected in the infants were not dominant in the mothers, but were still transmissible to the infants. As dense fractions are known to contain antibody-bound HCV particles, maternal antibodies against HCV may inhibit viral transmission. J. Med. Virol. 51:225–230, 1997. © 1997 Wiley-Liss, Inc.     

丙型肝炎病毒非结构区NS3基因在大肠埃希菌中的可诱导性表达

目的 构建丙型肝炎病毒（HCV）NS3基因的原核细胞表达载体。实现在大肠埃希菌中的可诱导性表达。方法 应用聚合酶链反应（PCR）技术，以美国HCV-H株全长cDNA质粒为模板，扩增获得NS3基因片段，克隆到原核表达载体pET-30C( )中，构建原核表达载体pET-NS3，转化BL21（DE3）宿主菌，以IPTG诱导，获得NS3蛋白的可诱导性表达，以HCVNS3的单链可变区抗体（ScFv)证实表达的NS3蛋白的特异性，结果 以HCVNS3基因序列特异性引物，PCR扩增获得1893bp的NS3DNA征段，插入pET-30C( )表达载体，转化BL21（DE3）受体菌，经培养，IPTG诱导，获得了重组HCVNS3蛋白的表达，以HCVNS3的ScFv证实了表达的重组蛋白HCVNS3的特异性。结论 以大肠埃希菌表达了HCVNS3的重组蛋白质。     

Hepatitis C virus envelope glycoprotein co-evolutionary dynamics during chronic hepatitis C

Hepatitis C virus (HCV) envelope glycoprotein co-evolution was studied in 14 genotype 1-infected and treatment-naive subjects, including 7 with mild and 7 with severe liver disease. Cassettes encoding the envelope 1 gene (E1) and hypervariable region (HVR1) of the envelope 2 gene were isolated at 38 different time points over 81 follow-up years. There were no significant differences in age, gender, alcohol use, or viral load between the mild and severe disease groups. Virus from subjects with severe disease had significantly slower evolution in HVR1, and significant divergent evolution of E1 quasispecies, characterized by a preponderance of synonymous mutations, compared to virus from subjects with mild disease. Phylogenetic comparisons indicated higher similarity between amino acid sequences of the E1 and HVR1 regions with mild disease versus severe disease (r=0.44 versus r=0.17, respectively; P=0.01). In summary, HCV envelope quasispecies co-evolution differs during mild versus severe disease.     

Hepatitis C virus-related resistance mechanisms to interferon alpha-based antiviral therapy.

Only 50-60% of the patients chronically infected with the hepatitis C virus (HCV) achieve a sustained virologic response to the current standard antiviral therapy consisting of pegylated interferon alpha in combination with ribavirin. The definite reasons for virologic response or non-response to interferon alpha-based therapy are unknown. Besides host and treatment efficacy factors, it is presumable that HCV is able to antagonize the antiviral activity of interferon alpha. So far, among the different HCV proteins, the envelope (E)2 protein, the non-structural (NS)3/4A protein, and the NS5A protein have been associated with interferon alpha resistance mechanisms in vitro. The clinical significance of amino acid mutations within these HCV proteins in HCV isolates from patients who did or did not respond to interferon alpha-based therapy was investigated in multiple studies. Within the E2 (HVR2, CD81 binding sites, PePHD) and the NS3/4A proteins no specific mutations in correlation with virologic response to interferon alpha-based therapy were observed. For the NS5A protein, mutations within the interferon sensitivity determining region (ISDR) and the complete NS5A protein may be of importance for response to interferon alpha-based treatment in patients infected with HCV subtype 1a/b.     

Sequence evolution and cross-reactive antibody responses to hypervariable region 1 in acute hepatitis C virus infection

Hepatitis C virus (HCV) infection may result in acute resolving or chronic infection. Patients that clear the infection have a more vigorous cellular immune response and an early humoral response to the hypervariable region 1 (HVR1) of the E2 envelope protein. To analyse further the properties of the early anti-HVR1 response, cross-reactivity of anti-HVR1 responses was assessed in five patients with acute HCV infection, who were infected by the same virus strain during a nosocomial outbreak. The sequence evolution of HVR1 was examined in sequential serum samples up to 37 months post infection. Peptides were synthesised corresponding to the obtained HVR1 sequences and unrelated HVR1 sequences, and antibody reactivity to the peptides in sequential sera was investigated by ELISA. The results suggest an association between specific gaps in humoral immunity and the HVR1 sequence evolution during early infection. Possible interpretations of this phenomenon include immune escape mechanisms or suppression of specific anti-HVR1 antibodies.     

Genotype distribution and comparison of the putative envelope region of hepatitis C virus from Korean patients

Comparative nucleotide sequence studies of the genomes of hepatitis C virus (HCV) revealed that there are at least 6 different genotypes of HCV. The prevalence of HCV genotypes among the patients with liver diseases in Korea was investigated using the polymerase chain reaction (PCR) for the NS5 region. In the 75 HCV RNA positive samples, two genotypes, type 1b and type 2a, were the major causative agents which accounted for 60% and 33% of infections respectively, while 7% could not be assigned a genotype by the methods used. The nucleotide sequences of cDNAs encoding the putative envelope proteins from 10 type 1b and 5 type 2a genotype samples were analyzed. Approximately 31–42% of the nucleotide sequences of type 1b samples examined differed from those of different genotypes, In the case of type 2a samples, 36–42% of the nucleotide sequences differed from those of different genotypes. The diversities of the amino acid sequences were the same or greater than those of the nucleotide sequences. Two hypervariable regions (HVR1 and HVR2) were recognized in both HCV genomes of genotypes 1b and 2a. However, the sequence divergence within the HVR2 region of genotype 2a was less than that of genotype 1b. © 1995 Wiley-Liss, Inc.     

Analysis of a non-structural gene reveals evidence of possible hepatitis C virus (HCV) compartmentalization

Viral diversity is a hallmark of hepatitis C virus (HCV) infection; however, only limited data are available regarding HCV variability in extrahepatic sites, and none have systematically compared diversity in non‐structural and structural genomic regions. Therefore, HCV diversity in the NS5B and envelope 1 (E1) hypervariable region 1 (HVR1) genes was evaluated in matched sera and peripheral blood mononuclear cells (PBMCs) obtained from 13 HCV‐infected women. Multiple clonal sequences were compared to evaluate quasispecies diversity and viral compartmentalization in PBMCs. Genetic distances were higher for E1/HVR1 compared to NS5B in both the sera and PBMCs (P = 0.0511 and 0.0284). Genetic distances were higher in serum NS5B compared to PBMC NS5B (P = 0.0003); however, they were not different when comparing E1/HVR1 in sera to PBMCs. By phylogenetic analysis of NS5B, evidence of possible PBMC compartmentalization was observed for one woman, while statistical methods were consistent with PBMC compartmentalization for six women. Evidence of compartmentalization within a non‐structural genomic region may suggest that viral adaptation to a unique extracellular microenvironment(s) may be required for efficient replication and could contribute to HCV persistence. J. Med. Virol. 84:242–252, 2012. © 2011 Wiley Periodicals, Inc.