SARS病毒核衣壳蛋白的表达与鉴定

依据Genebank中SARS基因组序列和酵母菌对密码子的选择性,采用人工合成的方法,合成了优化的SARS病毒核衣壳蛋白(N)的全基因(1296bp),与CTL表位基因(195bp)重组后,将其克隆到酵母分泌型表达载体pPIC9K中,构建成重组表达载体pPIC9K-N.重组载体转化毕赤酵母GS115,并经MD平板和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115-pPIC9K-N.用甲醇诱导其分泌表达目的蛋白并对表达产物进行分析、浓缩与鉴定.结果表明,SARS病毒核衣壳蛋白能实现在毕赤酵母中高效表达,表达量达到20%,初步纯化后的产物具有良好的抗原特异性.     

口蹄疫病毒3ABC基因截短体在毕赤酵母中的表达及鉴定

将长为525 bp的口蹄疫病毒3ABC基因截短体克隆到毕赤酵母表达载体pPIC9K中, 构建了重组表达质粒pPIC9K-3ABCt. 用BglⅡ线性化后, 电转化毕赤酵母菌GS115, 经表型筛选, PCR鉴定, 获得阳性重组菌(GS115/pPIC9K-3ABCt). 然后进行诱导表达, 通过SDS-PAGE和Western blot鉴定表达产物. 结果表明, 重组菌株成功分泌表达了分子量为40000, 具有免疫反应活性, 且呈二聚体形式的目的蛋白. 在96 h时表达量达到最高峰, 占分泌总蛋白的18%, 达到23.4 mg/L. 为进一步研制口蹄疫免疫和感染动物鉴别诊断试剂奠定了基础.     

人乳头瘤病毒HPV-16衣壳蛋白的结构特征

人乳头瘤病毒HPV-16是引发子宫颈癌的主要元凶.研究HPV-16病毒的结构特点,对于二十面体病毒几何结构的认识、描述和疫苗设计具有重要意义.综述了HPV-16病毒的衣壳结构特征、衣壳蛋白,分析了它们之间的相互作用,并试探性地指出了HPV-16衣壳几何结构的数学问题.     

模拟构筑二十面体病毒衣壳结构的研究进展

许多病毒具有二十面体对称结构,二十面体病毒衣壳结构的模拟构筑已成为物质结构的研究目标之一.介绍了二十面体病毒衣壳结构的几种模型,对各种模型的特点及应用范围进行了归纳,并对衣壳结构模拟构筑的意义进行了简要介绍.     

仿病毒衣壳自组装体及其生物医学应用

仿病毒衣壳结构自组装体具有重要的基础研究和应用价值,是化学、材料、生物医学等多学科前沿交叉领域.本文从天然病毒衣壳的基本结构和特征出发,立足于从结构仿生到功能仿生的角度,综述了以天然病毒衣壳蛋白质和人工合成材料为组装基元构建仿病毒衣壳自组装体的策略,及其形态结构的调控和功能优化等.重点论述了近年来合成肽类分子在仿病毒衣壳自组装体结构和功能方面的进展.同时,就仿病毒衣壳自组装体在药物控释、基因传递等生物医学领域的应用也做了论述.     

猪IL-18成熟蛋白在大肠杆菌中的表达及生物学活性鉴定

以含猪IL-18全基因的重组质粒pGEM-IL-18为模板,PCR扩增猪IL-18成熟蛋白基因.将IL-18成熟蛋白片段定向插入原核表达载体pET-28a(+)中,构建重组表达质粒pET-IL-18,转化大肠杆菌BL21(DE3),在IPTG诱导下表达融合蛋白(His-IL-18),并进行融合蛋白的纯化、生物学活性鉴定.结果表明,SDS-PAGE可检测到相对分子质量约为2.1×104的融合蛋白,westem blot证实His-IL-18能与猪IL-18单克隆抗体发生特异性反应.重组猪IL-18经纯化后,能明显刺激猪脾脏T淋巴细胞增殖反应,在Marc-145细胞上抗猪繁殖与呼吸综合征病毒的活性为2.50×103IU/mg,在PK-15细胞上抗猪伪狂犬病毒、猪细小病毒的活性分别为2.00×103和2.24×103IU/mg.表明建立的表达系统能够表达重组猪IL-18,表达的重组猪IL-18具有一定的生物学活性.     

Expression and Characterization of a Recombinant Truncated Capsid Protein of Hepatitis E Virus in Pichiapastoris

Hepatitis E is an enterically transmitted viral disease caused by infection with hepatitis E virus(HEV).HEV is a nonenveloped virus that has been classified in the family of Caliciviridae.The virus appears to be a polyadenylated,positive-stranded RNA virus with three major open reading frames(ORFs).The capsid protein of HEV is encoded by the open reading frame 2(ORF2).We attempted to produce a truncated capsid protein,designed p293,in Pichia pastoris.The p293 gene encoding amino acids(aa) 382-674 of HEV ORF2 was designed based on the full length of HEV ORF2,cloned into the yeast vector pPIC9K,and expressed in P.pastoris strain GS115.SDS-PAGE and Western blotting demonstrated that the recombinant protein p293 could well be expressed in P.pastoris.Under optimized conditions (culture medium pH,6.0―6.5;methanol concentration added daily,3.0%;inoculum density,OD600=60;induction time point,72―96h),the yield of soluble p293 was approximately 80 mg/L.We also observed p293 secretory expressed in P.pastoris to be 30 nm viral like particles by using electron microscopy.These results show that the p293 may has utility in the analysis of cell specific factors in the protein processing and assembly of HEV,and serve as a useful antigen for both diagnostic and vaccine purposes.     

靶向喉癌的蜂毒肽重组抗体的构建及鉴定

利用大肠杆菌表达系统制备了重组融合蛋白antiEGFR/MEL,并用Ni2+层析柱对其进行了纯化.该重组蛋白中抗表皮生长因子受体(antiEGFR)单链抗体(scFv)主要靶向喉癌细胞中的EGFR,而蜂毒肽(MEL)主要抑制肿瘤细胞增殖.采用SDS-PAGE和Westernblot检测证明了antiEGFR/MEL的有效表达.共聚焦显微镜和流式细胞术实验结果表明,antiEGFR/MEL可与Hep-2肿瘤细胞有效结合,而几乎不与EGFR阴性的Jurkat细胞结合.噻唑蓝(MTT)检测结果说明,antiEGFR/MEL可有效抑制人喉癌细胞Hep-2的增殖.以上结果表明,antiEGFR/MEL能够有效靶向EGFR阳性肿瘤细胞,并有效抑制肿瘤细胞增殖,有望应用于EGFR靶向肿瘤治疗.     

乙型肝炎病毒表面抗原preS基因在痘苗病毒系统中的分泌型表达

本文对带preS2的乙肝病毒表面抗原基因以及带preS1和preS2的乙肝表面抗原全基因的重组痘苗病毒vTMS-1和vTLS-1的表达和分泌进行了研究。在人TK-143细胞中它们能表达各自所编码的蛋白——乙肝表面抗原中分子蛋白和大分子蛋白。产物是可分泌的,并能形成22nm颗粒。分泌在培液中的表达产物能分别与抗preS2和抗preS1的单抗反应,并保持了天然表面抗原大分子和中分子蛋白所具有的多聚人血清白蛋白的受体活力。它们的表达产物中都还合有乙肝表面抗原主蛋白的成分。     

截短与修饰的β-淀粉样蛋白与阿尔茨海默病

β-淀粉样蛋白(Aβ)的聚集和沉积被认为是导致阿尔茨海默病的重要因素,早前的研究多集中在全长无修饰的Aβ_1-40和Aβ_1-42上。近些年研究发现,在AD患者大脑内存在着多种截短与修饰的Aβ蛋白,它们对AD的疾病进程有不可忽视的贡献。例如,焦谷氨酸Aβ、磷酸化Aβ被认为是AD患者出现症状的标志;截短的Aβ_4-40/42在患者脑内的含量与Aβ_1-40/42接近且具有类似的聚集性质和毒性;患者脑内氧化压力升高导致的酪氨酸硝基化、二聚化和甲硫氨酸氧化形式的Aβ也具有不同的性质。本文对这些截短与修饰的Aβ蛋白的产生、结构、毒性以及和AD的关联进行了综述。     

Current Progress in the Development of Hepatitis B Virus Capsid Assembly Modulators: Chemical Structure,Mode-of-Action and Efficacy

Hepatitis B virus (HBV) is a major causative agent of human hepatitis. Its viral genome comprises partially double-stranded DNA, which is complexed with viral polymerase within an icosahedral capsid consisting of a dimeric core protein. Here, we describe the effects of capsid assembly modulators (CAMs) on the geometric or kinetic disruption of capsid construction and the virus life cycle. We highlight classical, early-generation CAMs such as heteroaryldihydropyrimidines, phenylpropenamides or sulfamoylbenzamides, and focus on the chemical structure and antiviral efficacy of recently identified non-classical CAMs, which consist of carboxamides, aryl ureas, bithiazoles, hydrazones, benzylpyridazinones, pyrimidines, quinolines, dyes, and antimicrobial compounds. We summarize the therapeutic efficacy of four representative classical compounds with data from clinical phase 1 studies in chronic HBV patients. Most of these compounds are in phase 2 trials, either as monotherapy or in combination with approved nucleos(t)ides drugs or other immunostimulatory molecules. As followers of the early CAMs, the therapeutic efficacy of several non-classical CAMs has been evaluated in humanized mouse models of HBV infection. It is expected that these next-generation HBV CAMs will be promising candidates for a series of extended human clinical trials.     

分子信标用于不对称PCR检测乙型肝炎病毒

为了缓解竞争杂交对分子信标检测的影响，将分子信标与不对称聚合酶链式反应(PCR)联用进行血清中乙型肝炎病毒(HBV)的检测。并采用更为直接的荧光方法，将不对称PCR扩增中的引物浓度比确定为5：1，该结果与电泳方法得到的结果有所不同。文中结合对称及不对称PCR过程中荧光强度的变化情况，对其原因进行了详细的探讨。分子信标与不对称PCR联用，可以专一地检测出血清中HBV的存在，与分子信标／对称PCR相比．其检测效果明显增强。     

Expression of Human Hepatitis B Virus Surface Antigen Gene in Transgenic Tobacco

Expression of Human hepatitis B virus surface antigen (HBsAg) gene in plant was reported for the first time. The recombinant plasmid pRoKⅡ-HBsAg was constructed by inserting HBsAg gene into the downstream of CaMV 35S promoter of binary vector pRoKⅡ and then introduced into Agrobacterium tumefaciens LBA4404. The kanamycin-resistant plants were obtained by Agrobacterium-mediated transformation system. It was shown that HBsAg gene was expressed in transgenic tobacco plants and their progenies by ELISA. The spherical particles of ψ 22 nm in the leaf extract of trangenic tobacco were observed by immunosorbent electron microscopy.     

Expression Optimization and Characterization of the Catalytic Domain of Human MT3-MMP

Introduction Matrixmetalloproteinases(MMPs)areafamilyof calciumandzincrequiringendoproteinasesthattogether candegradeallthemaincomponentsoftheextra cellu larmatrixandbasementmembranes[1].MMPsarein volvedinawiderangeofproteolyticevents,innormal andpatholog…     

Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase

Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5′-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33–353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg²⁺) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg²⁺ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.     

Prokaryotic Expression, Purification, Antibody Production of a NH2 -Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

mCLCA3 is a member of calcium activated chloride channel(CACC)family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung.To study the protein structure and expression of mCLCA3 in asthmatic mouse lung,an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E.coli,purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated.Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen.Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3.The results demonstrate that the 90 kDa N-terminal peptide,neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.     

Isolation and Purification of Recombinant HBsAg of Human Hepatitis B Virus from Silkworm Larvae

Recombinant HBsAg coded by preS1-preS2-S regions of hepatitis B virus was expressed in Bombyx mori silkworm larvae. Recombinant HBsAg was expressed (30–40 μg/mL, 0.1% of the total amount of extracted protein) by larvae infected with recombinant baculovirus rBmNPV-Hep-preS1-S containing cDNA of HBsAg strictly by the polyhedrin gene promoter. Recombinant HBsAg consisting of a polypeptide of molecular weight ∼36 kDa (p36) was purified by gel filtration and affinity chromatography to 92% purity. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 477–480, September–October, 2005.