Mapping of Homologous Interaction Sites in the Hepatitis B Virus Core Protein

Hepatitis B virus consists of an outer envelope and an inner capsid, or core, that wraps around the small genome plus the viral replication enzyme. The icosahedrally symmetric nucleocapsid is assembled from multiple dimeric subunits of a single 183-residue capsid protein, which must therefore contain interfaces for monomer dimerization and for dimer multimerization. The atomic structure of the protein is not known, but electron microscopy-based image reconstructions suggested a hammerhead shape for the dimer and, very recently, led to a tentative model for the main chain trace. Here we used a combination of interaction screening techniques and functional analyses of core protein variants to define, at the primary sequence level, the regions that mediate capsid assembly. Both the two-hybrid system and the pepscan technique identified a strongly interacting region I between amino acids (aa) 78 and 117 that probably forms part of the dimer interface. Surprisingly, mutations in this region, in the context of a C-terminally truncated but assembly-competent core protein variant, had no detectable effect on assembly. By contrast, mutations in a second region, bordered by aa 113 and 143, markedly influenced capsid stability, strongly suggesting that this region II is the main contributor to dimer multimerization. Based on the electron microscopic data, it must therefore be located at the basal tips of the dimer, experimentally supporting the proposed main chain trace.     

乙型肝炎病毒聚合酶基因B区和C区序列的异质性

乙型肝炎病毒(HBV)聚合酶基因编码约816个氨基酸的多蛋白,其氨基端为末端蛋白,羧基端为RNA酶H,后者上游紧邻反转录酶/DNA聚合酶.通过计算机模拟分析将HBV聚合酶基因分为假想的A到E共5个区段.     

一例乙型肝炎患者体内不同HBV克隆的序列差异

采用ＰＣＲ扩增、噬菌体克隆和核苷酸序列分析的方法对一例乙型肝炎患者体内ＨＢＶ的ＨＢｓＡｇ编码区和部分前Ｓ２区进行序列分析。结果发现同一患者体内获得的不同ＨＢＶＤＮＡ克隆间存在个别碱基的差异,而这种差异并非方法学本身产生的。由此得出的结论是,这种碱基“突变”反映了乙型肝炎患者体内ＨＢＶ基因组实际存在的多态性。     

一例乙型肝炎患者体内不同HBV克隆的序列差异

采用PCR扩增、噬菌体克隆和核苷酸序列分析的方法对一例乙型肝炎患者体内HBV的HBsAg编码区和部分前S2区进行序列分析.结果发现同一患者体内获得的不同HBV DNA克隆间存在个别碱基的差异,而这种差异并非方法学本身产生的.由此得出的结论是,这种碱基"突变"反映了乙型肝炎患者体内HBV基因组实际存在的多态性.     

犬瘟热病毒RT-LAMP检测方法的建立和初步应用

目的利用环介导等温扩增（LAMP）技术建立一种检测犬瘟热病毒感染的新方法。方法根据GenBank中NP基因序列,设计4条LAMP特异性引物,对反应条件、特异性、可视化效果和应用效果进行研究。结果在60℃等温的条件下、1 h内可完成RT-LAMP扩增过程;特异性和可视效果良好;对63份临床标本进行检测,阳性检出率为71.4%（45/63）,检出率高于RT-PCR的63.5%（40/63）。结论建立的RT-LAMP检测方法,显示了较高的特异性和敏感性,而且兼具高效、快捷、可视化的优势,为临床检测犬瘟热病毒感染提供了一种快速简便的新途径。     

乙型肝炎病毒RT区变异及其与基因型分布的关系

目的：探讨大样本乙型肝炎病毒（HBV）感染患者RT区耐药位点变异的流行情况,及各耐药位点变异与HBV基因型的关系。方法：采用P区测序法对1117例慢性乙型肝炎患者的血清病毒进行P区测序、进化树分型。结果：RT区耐药位点变异发生率与基因型关系密切,在基因型C患者中的变异发生率远远高于基因型B患者（P=O．000）。Rt180、rtM204V、rtM204I、rt181、rt213位点变异均与基因型c有关（P〈O．05）。主要的三种变异类型rt180＋rtM204V、rtM204I、rt180＋rtM204I间基因型分布存在显薯差异（P=0．003）。不同HBeAg状态下,耐药变并的发生有显著差并（P=O．020）,特别是rt181和rt236位点变畀。结论：HBV基因型影响RT区耐药变异发生率及变异类型。且耐药变异发生率也与HBeAg状态有关。     

Evidence for the Involvement of the Small Subunit of HIV-1 Reverse Transcriptase (RT) in the TSAO-Resistance

Abstract

By introducing the TSAO-resistance mutation 138Glu→Lys in only the p51 subunit of HIV-1 RT, we obtained compelling evidence for a structural and functional role of the p51 subunit in the sensitivity and/or resistance of the enzyme to the TSAO-derivatives.     

人端粒逆转录酶与Snapin蛋白相互作用的鉴定

目的:研究人端粒逆转录酶(hTERT)与Snapin蛋白的相互作用。方法:将hTERT基因和Snapin基因分别构建到pGBKT7和pGADT7载体中,用酵母双杂交方法在酵母中验证其相互作用;在293T细胞中,共转染带有Flag标签的hTERT及带有GFP标签的Snapin质粒,进行免疫共沉淀;将GST融合的Snapin纯化蛋白与hTERT进行GST-pull down,验证其相互作用。结果:3种方法都证明hTERT能够与Snapin相互作用。结论:Snapin蛋白能够与hTERT相互作用,为研究Snapin参与调控端粒酶的功能活动提供了一些线索。