重组人巨细胞病毒(HCMV)嵌合肽在毕赤酵母中的表达

目的：在巴期德毕赤酵母中表达人巨细胞病毒gp52C末端和pp150C末端串联片段的嵌合肽，方法：用SacI和BglⅡ分别酶切CMVp-pPIC9K重组质粒，电打孔法导入毕赤酵母GS115后，在缺组氨酸的MD板上筛选出转化子，然后根据甲醇利用快速型（Mut^ ）和甲醇利用缓慢型(Mut^s）菌株的不同生长特点，筛选出Mut^ 和Mut^s型转化子，用PCR法进一步鉴定阳性克隆，分别用甲醇诱导Mut^ 和Mut^s型转化子表达目的蛋白4d，取培养产物冻干浓缩，进行SDS-PAGE和Western blotting，筛选出能特异表达目的的蛋白的菌株，分析蛋白的含量及纯度。结果：重组人巨细胞病毒可在甲醇利用快速型毕赤酵母中有效表达，其表达量约占培养上清分泌蛋白的76.5%，结论：重组人巨细胞病毒（HCMV）嵌合肽可在真核细胞毕赤酵母中成功表达。     

The UFM1 Pathway Impacts HCMV US2-Mediated Degradation of HLA Class I

To prevent accumulation of misfolded proteins in the endoplasmic reticulum, chaperones perform quality control on newly translated proteins and redirect misfolded proteins to the cytosol for degradation by the ubiquitin-proteasome system. This pathway is called ER-associated protein degradation (ERAD). The human cytomegalovirus protein US2 induces accelerated ERAD of HLA class I molecules to prevent immune recognition of infected cells by CD8⁺ T cells. Using US2-mediated HLA-I degradation as a model for ERAD, we performed a genome-wide CRISPR/Cas9 library screen to identify novel cellular factors associated with ERAD. Besides the identification of known players such as TRC8, p97, and UBE2G2, the ubiquitin-fold modifier1 (UFM1) pathway was found to affect degradation of HLA-I. UFMylation is a post-translational modification resembling ubiquitination. Whereas we observe ubiquitination of HLA-I, no UFMylation was detected on HLA-I or several other proteins involved in degradation of HLA-I, suggesting that the UFM1 pathway impacts ERAD in a different manner than ubiquitin. Interference with the UFM1 pathway seems to specifically inhibit the ER-to-cytosol dislocation of HLA-I. In the absence of detectable UFMylation of HLA-I, UFM1 may contribute to US2-mediated HLA-I degradation by misdirecting protein sorting indirectly. Mass spectrometry analysis of US2-expressing cells showed that ribosomal proteins are a major class of proteins undergoing extensive UFMylation; the role of these changes in protein degradation may be indirect and remains to be established.     

桥序列在构建人工核酶M1GS中的作用

目的：为检验连接的Gs序列是否会影响M1RNA高级结构而导致M1RNA活性改变,对核酶的催化机制进行探讨。方法：通过软件模拟对M1GS进行结构分析,筛选了一段独立折叠的桥序列连接M1RNA与GS,并通过体外切割实验检验有桥和无桥序列的核酶的活性。结果：有桥序列的M1GS具备体外特异切割底物的核酶活性,而无桥序列的M1GS核酶未表现体外切割活性。结论：证实桥序列对维持M1RNA高级结构和保持人工核酶M1GS的体外切割活性具有重要作用。     

噻唑烷-4-酮衍生物抗HCMV和VZV的活性

利用病毒噬斑抑制法,探讨了2,3-二芳基-1,3-噻唑烷-4-酮(化合物1和2)、含糖基的1,3-噻唑烷-4-酮(化合物3和4)抗人巨细胞疱疹病毒(HCMV)和水痘带状疱疹病毒(VZV)活性.结果表明:2,3-二芳基-1,3-噻唑烷-4-酮是一类新型高效的抗HCMV化合物,而含糖基的1,3-噻唑烷-4-酮可能是一类比较...     

用PCR法检测人巨细胞病毒DNA

用聚合酶链式反应（ＰＣＲ）法检测患者尿液中人巨细胞病毒（ＨＣＭＶ）ＤＮＡ．结果表明，自行设计合成的引物位于ＨＣＭＶ基因组早期蛋白基因区，经ＰＣＲ仪扩增一段长４３０ｂｐ的特异序列片段，对正常人基因组ＤＮＡ或其它疱疹病毒ＤＮＡ无交叉反应．此法可检测出少至１０－１６ｇ（０．１ｆｇ）的病毒ＤＮＡ．通过对３５份尿液标本的检测，比较ＰＣＲ法和组织培养法的检测结果完全一致     

Drug Discovery of Nucleos(t)ide Antiviral Agents: Dedicated to Prof. Dr. Erik De Clercq on Occasion of His 80th Birthday

Nucleoside and nucleotide analogues are essential antivirals in the treatment of infectious diseases such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV). To celebrate the 80th birthday of Prof. Dr. Erik De Clercq on 28 March 2021, this review provides an overview of his contributions to eight approved nucleos(t)ide drugs: (i) three adenosine nucleotide analogues, namely tenofovir disoproxil fumarate (Viread^®) and tenofovir alafenamide (Vemlidy^®) against HIV and HBV infections and adefovir dipivoxil (Hepsera^®) against HBV infections; (ii) two thymidine nucleoside analogues, namely brivudine (Zostex^®) against HSV-1 and VZV infections and stavudine (Zerit^®) against HIV infections; (iii) two guanosine analogues, namely valacyclovir (Valtrex^®, Zelitrex^®) against HSV and VZV and rabacfosadine (Tanovea^®-CA1) for the treatment of lymphoma in dogs; and (iv) one cytidine nucleotide analogue, namely cidofovir (Vistide^®) for the treatment of HCMV retinitis in AIDS patients. Although adefovir dipivoxil, stavudine, and cidofovir are virtually discontinued for clinical use, tenofovir disoproxil fumarate and tenofovir alafenamide remain the most important antivirals against HIV and HBV infections worldwide. Overall, the broad-spectrum antiviral potential of nucleos(t)ide analogues supports their development to treat or prevent current and emerging infectious diseases worldwide.     

TaqMan技术快速定量检测人巨细胞病毒的早期感染

采用实时TaqMan技术快速定量检测血清中的人巨细胞病毒(HCMV)感染,在HCMV保守性的Major IE基因区段设计了一对引物和一条Taqman探针,在LightCycler及Rotor-Gene定量检测仪上检测HCMV DNA.试验证明,两种仪器定量结果具有一致性,以PCR模板浓度来定义,敏感度达到1.0×102拷贝/μL,线性范围为1.0×102～1.0×108拷贝/μL.应用该方法与ELISA法对40例临床婴幼儿患者血清同时进行HCMV检测,结果显示TaqMan法与ELISA法相关性不高,前者更适于HCMV感染早期的定量检测,提示该法可应用于孕妇及新生儿HCMV筛查,并有助于疗效监测和评估.     

核酶M1GS-T6对HCMV UL97基因RNA片段体外切割作用

目的:研究M1GS核酶对HCMV UL97mRNA的体外切割作用.方法:针对HCMV UL97 mRNA T6位点设计与之互补的引导序列(Guide Sequence,GS),将其共价结合至大肠杆菌核酶P催化亚基(M1 RNA)的3'末端,构建M1GS-T6核酶,并用其对UL97基因亚克隆片段转录产物进行体外靶向切割实验.结果:核酶M1GS-T6具备特异性切割靶分子UL97 mRNA的能力.结论:核酶M1GS-T6具备特异性切割活性,为进一步研究HCMV病毒基因功能和治疗提供了新的途径.     

Durumolides A-E, anti-inflammatory and antibacterial cembranolides from the soft coral Lobophytum durum

Chemical investigations on the acetone extract of the soft coral Lobophytum durum have afforded five new cembranoids with a trans-fused α-methylene-γ-lactone, durumolides A-E (1, 6, and 8-10), and five previously characterized cembrane-based diterpenoids (2-5 and 7). The structures of the isolated metabolites were elucidated through extensive spectroscopic analyses, while the relative stereochemistry of 4 was confirmed by X-ray diffraction analyses. Moreover, the absolute configurations of 3-5 and 8 were established by application of modified Mosher's method. The anti-inflammatory effects, antibacterial activities, and inhibition assay of HCMV (Human cytomegalovirus) endonuclease activity of these isolated metabolites 1-10 were evaluated in vitro.