新型抗HIV-1蛋白GRFT的构建、表达及活性研究

根据已知的新型抗HIV-1蛋白GRFT基因氨基酸序列,推测其DNA编码序列,密码子优化及修饰后进行全基因化学合成,连接到原核表达载体pET28a（+）中,转化大肠杆菌BL21(DE3),IPTG诱导表达,获得目的蛋白. SDS-PAGE、Western Blot分析结果表明,目的蛋白得到良好表达并具有抗原活性;对表达条件进行优化,在最佳表达条件下,目的蛋白的表达量可占菌体总蛋白的55.84%;利用Ni2+-NTA柱亲和层析法进行目的蛋白的复性和纯化,凝胶成像系统扫描分析表明,纯度可达94.06%;运用Dot-ELISA法进行复性蛋白的抗原结合活性检测,结果显示,目的蛋白能够与HIV-1膜蛋白抗原特异性结合,初步证明所表达的重组蛋白具有良好的体外结合活性;基于制备的能够表达HIV-1 gp120基因的靶细胞模型,运用IFA法开展目的蛋白的细胞结合活性研究,结果显示,目的蛋白能够与靶细胞发生特异性反应,表明成功制备并获得了具有生物活性的新型抗HIV-1蛋白GRFT,为进一步研制新型抗HIV-1基因工程药物及其靶向治疗制剂奠定了坚实基础.     

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.     

重组人OCIF结构域D1～D6基因在毕赤酵母中的表达

利用PCR以实验室构建的原核重组表达质粒pProEX-OCIF为模板扩增得到N末端融合有6×His标签和rTEV蛋白酶识别序列的人破骨细胞形成抑制因子(OsteoclastogenesisInhibitoryFactor,简称OCIF)结构域D1～D6(简称O CIFm)编码基因片段;将其与pMD18-T连接,转化大肠杆菌TOP10,筛选得到阳性重组质粒pMD18-OCIFm,双酶切重组克隆质粒pMD18-OCIFm得到OCIFm基因片段;将其定向插入甲醇营养型酵母分泌表达载体pPIC9中,构建获得重组表达质粒pPIC9-OCIFm.测序验证后,以限制性内切酶SalⅠ线化,电穿孔转化酵母宿主菌GS115.筛选得到阳性表达菌株后,甲醇诱导表达4d,SDS-PAGE和Westernblot对表达情况进行分析和确认.所获得的OCIFm基因片段在甲醇营养型酵母中表达量占菌体总蛋白的30%以上.利用Ni-NTA树脂对表达产物进行一步亲和层析纯化.活性测定表明纯化的表达产物可诱导体外培养的成熟破骨细胞样细胞的凋亡.表达产物的生物学活性较利用原核表达系统明显提高.     

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.     

HIV-1外膜蛋白在毕赤酵母中的构建

为了进一步研究HIV-1外膜蛋白的结构和功能,获得足够量的糖蛋白,对HIV-1的外膜蛋白Env进行了修饰,利用PCR技术克隆目的基因,把Env基因构建到毕赤酵母Pichia pastoris表达载体中,把pPICZαB-Env表达载体转化到酵母中,根据基因重组技术,使Env基因和酵母基因组重组,筛选阳性重组子.     

肠道病毒71型外壳蛋白VP2在Pichia.pastoris酵母中的表达

利用逆转录聚合酶链式反应(RT-PCR)的方法,克隆肠道病毒71型(Enterovirus 71,EV71)SHZH03株外壳蛋白VP2基因,构建酵母分泌型表达质粒pPIC9K/VP2,经序列测定证实α-factor信号肽和VP2的序列和阅读框正确后,用SacI酶切使之线性化,电转化法将目的基因整合到宿主菌GS115基因组上;经转化子表型筛选和PCR分析鉴定后,甲醇诱导筛选到高效表达VP2蛋白的工程菌株.用饱和硫酸铵法对重组蛋白VP2进行了较好的纯化.Western Blot分析表明,重组蛋白VP2具有较好的反应原性,从而为发展EV71快速、高效、廉价诊断试剂奠定了基础.     

人三叶因子3双结构域突变体的构建

利用基因工程方法在大肠杆菌-酶母穿梭质粒pPIC9K上构建了人三叶因子3(HumanTrefoilFactor3,hTFF3)双结构域突奕体基因,采用毕氏酵母表达系统对目的基因进行了分泌表达。经S-Sepharose,Q-Sepharose,SephacrylS-100纯化后得到突变体蛋白,主要以单体形式存在。SDS-PAGE表明重组蛋白分子量约为12000,并在Westernblotting印迹实验中证明能被抗hTFF3抗体所识别,N-端氨基酸测序与天然hTFF3-一致,质谱检测纯化的蛋白出现单体和双体两种形式。用重组蛋白对乙醇诱导的大鼠胃溃疡进行实验治疗表明hTFF3双体突变体与天然形成的双体蛋白具有相同的活力,并且活力高于单体形式的hTFF3。     

抗肿瘤双特异免疫导向治疗制剂CAtin的表达及活性分析

结合生物信息学方法与已知癌胚抗原(Carcinoembryonic antigen, CEA)特异性单链抗体(Single chain Fv fragment, scFv)核苷酸序列, 经分子设计和密码子优化后, 通过化学方法合成CEA二硫键稳定性单链抗体(Disulfide stabilized single chain Fv fragments, scdsFv)基因片段. 将凋亡素基因(Apoptin)通过一段柔性连接肽(Linker)连接在CEA scdsFv基因下游, 并克隆入大肠杆菌表达载体质粒pET28a, 转化BL21感受态菌后经异丙基-β-D-硫代半乳糖苷(Isopropyl β-D-1-thiogalactopyranoside, IPTG)诱导, 表达融合蛋白CAtin. SDS-PAGE和Western-Blot分析表明, 目的蛋白得到良好表达, 经条件优化后表达量最高可达44.1 mg/L. 融合蛋白经分步洗涤法和谷胱甘肽对表达的目的蛋白进行初步纯化和复性后, 利用人肝癌细胞(HCC)对所制备融合蛋白进行亲和力测定、细胞结合活性测定和特异性细胞杀伤活性分析. 结果显示, 所制备融合蛋白不仅能够有效地与上述肿瘤细胞结合, 并对其具有明显的杀伤活性, 表明成功制备了具有特异性识别和特异性杀伤活性的双特异抗肿瘤免疫导向制剂.     

用作HIV-1融合抑制剂的多肽及其类似物

HIV-1通过其包膜糖蛋白跨膜亚基gp41介导的病毒-细胞膜融合进入和感染靶细胞.HIV-1融合抑制剂以gp41为靶点,通过阻断病毒与宿主细胞膜的融合,在感染的初始环节切断HIV-1的复制周期.2003年,首个多肽类融合抑制剂T-20获美国食品药物管理局（FDA）批准上市,但其易被体内蛋白酶降解、临床剂量大、耐受性差,且耐药性HIV-1毒株也很快出现.针对这些缺点,近年来在融合抑制剂的作用机制研究和新融合抑制剂的研发等方面取得了重要进展.以gp41不同功能区为靶点,具有高活性和更好代谢性质的多肽及多肽类似物候选分子不断被发现,成为抗HIV药物研究领域的热点之一.本文综述了多肽和类肽类融合抑制剂的研究进展,为相关的药物开发和基础研究提供参考.     

肝素与HIV-1膜表面蛋白gp120相互作用的预测和模拟

用分子模拟软件研究肝素与HIV-1膜表面蛋白gp120的相互作用.将肝素中的单糖、二糖和三糖片段作为探针对gp120蛋白进行搜索,统计分析确定肝素结合区域.用肝素六糖片段和结合区域进行反应分子对接,获得两种结合模式.最终建立的模型能够很好地解释肝素体外抑制HIV-1的现象,同时对其机理进行了推测.     

Discovery of novel anti-HIV active G-quadruplex-forming oligonucleotides

A series of d((5')TGGGAG(3')) sequences, 5'-conjugated with a variety of aromatic groups through phosphodiester linkages, were synthesized, showing CD spectra diagnostic of parallel-stranded, tetramolecular G-quadruplex structures. When tested for anti-HIV-1 and HIV-2 activity, potent inhibition of HIV-1 infection in CEM cell cultures was found, associated with high selectivity index values. Surface Plasmon Resonance assays revealed specific binding to HIV-1 gp120 and gp41.     

Purification of a recombinantly produced transmembrane protein (gp41) of HIV I

The transmembrane protein gp41, a component of the viral envelope of HIV I, and its analogue gp36 of HIV II are important antigens for the sensitive and specific detection of anti-HIV antibodies. The immunodominant region of the protein gp41, which reacts with 100% of sera of infected persons, was produced by gene technological means in Escherichia coli. The protein accumulates in the form of insoluble inclusion bodies in the bacterial cell. Purification strategies for this aggregated material depend mainly on the isolation of these "inclusion bodies" and subsequent washing procedures. Growth conditions of the recombinant E. coli cells and the method of the cell disruption are important for the efficiency of purification and the recovery of the antigen. Owing to the insolubility of the expressed antigen, a significant concentration of recombinant gp41 was possible by extracting the soluble cell components. For this purpose, mild detergent solutions and low-molarity chaotropic buffer solutions were used. After final solubilization in 8 M urea buffer at pH 12.5, further chromatographic purification steps followed. The reduction of disulphide bridges with beta-mercaptoethanol or dithiothreitol was important. Gel filtration on a Sephacryl S-200 or Superose 12 column and/or ion-exchange chromatography on a DEAE-Sepharose Fast Flow or Mono Q HR (5/5) column finally resulted in the desired purity of the antigen.     

毕赤酵母表达的HIV-2外膜糖蛋白的纯化与活性测定

人免疫缺陷病毒(Human immunodeficiency virus,HIV)是艾滋病（AIDS）的病原体,其外膜蛋白是HIV侵入人体靶细胞的物质基础,对其结构与功能的深入研究不但能为彻底揭示HIV致病机理奠定基础,而且有助于抗感染免疫研究和治疗物的设计^[1,2],HIV－2ROD外膜蛋白gp105是一种高度糖基化的糖蛋白,近年来发展迅速的毕赤酵母（Pichia Pastoris）真核表达系统对分泌蛋白的糖基化修饰形式与天然的gp105相同^[3]。为实现大量制备有活性的gp105,我们用PCR法去除了gp105基因5′端28个密码子的非功能片段,以提高gp105的表达水平,并成功地在毕赤酵母中实现了gp105的高效分泌表达。本文对这一表面产物的纯化、性质及活性进行了研究,为尽快研制出有效的疫苗奠定基础。     

Research on anti-HIV-1 agents. Investigation on the CD4-Suradista binding mode through docking experiments

Sulfonated distamycin (Suradista) derivatives exhibit anti-HIV-1 activity by inhibiting the binding of the viral envelope glycoprotein gp120 to its receptor (CD4). With the aim to propose a possible binding mode between Suradistas and the CD4 macromolecule, molecular docking experiments, followed by energy minimization of the complexes thus obtained, were performed. Computational results show that ligand binding at the CD4 surface involves two or three positively charged regions of the macromolecule, in agreement with the results of X-ray crystallographic analysis of a ternary complex (CD4/gp120/neutralizing antibody) recently reported in the literature. Our findings account well for the structure–activity relationship found for Suradista compounds.     

Differential display identifies genes in chinese hamster ovary cells sensitive to elevated ammonium

Ammonium is a toxic waste product that has been reported to negatively inhibit cell growth and recombinant glycosylation in Chinese hamster ovary (CHO) cells; however, the effect of this toxicity on intracellular gene expression has received only limited investigation. We used a differential display method to identify genes in CHO cells that were affected by ammonium stress. Eight genes whose mRNA levels significantly changed in response to elevated ammonium were isolated and identified. Five of the genes were identified as having lower expression under the ammonium stress, whereas three genes were identified as having higher expression. Sequence homology with other mammalian organisms was used to attribute function to these newly identified genes. The identified ammonium-sensitive genes were grouped into three broad functional groups: cellular processes, energy metabolism, and genetic-information processing. The three cellular process-related genes had lower expression (anaphase-promoting complex subunit 5, eukaryotic initiation factor 5A II, KIAA1091 protein). The two energy-related genes had higher expression under ammonium stress (adenosine triphosphate synthase subunit C and mitofusin 1). Both of the genetic information-processing genes (endoplasmic reticulum [ER]-resident protein ERdj5 and structure-specific recognition protein 1) had lower expression under the ammonium stress, whereas the 26S proteasome subunit adenosine triphosphatase 3 gene had higher expression. These preliminary results indicate that ammonium stress lowers expression of genes controlling cell cycle, protein folding, and quality and raises genes that control energy metabolism and degradation. Our findings demonstrate the usefulness of mRNA differential-display techniques for the detection of CHO cell genes affected by ammonium stress.     

A combination of molecular dynamics and docking calculations to explore the binding mode of ADS-J1, a polyanionic compound endowed with anti-HIV-1 activity

The HIV-1 entry process is an important target for the design of new pharmaceuticals for the multidrug therapy of AIDS. A lot of polyanionic compounds, such as polysulfonated and polysulfated, are reported in the literature for their ability to block early stages of HIV-1 replication. Several studies have been performed to elucidate the mechanism of the anti-HIV-1 activity of sulfated polysaccharides and polyanions in general, including binding to cell surface CD4 and interfering with the gp120-coreceptor interaction. Here, we show molecular modeling investigations on ADS-J1, a polyanionic compound with anti-HIV activity that is able to interfere with gp120-coreceptor interactions. Agreeing with experimental data, computer simulations suggested that the V3 loop of gp120 was the preferential binding site for ADS-J1 onto HIV-1. Moreover, mutations induced by the inhibitor significantly changed the stereoelectronic properties of the gp120 surface, justifying a marked drop in the affinity of ADS-J1 toward an ADS-J1-resistant HIV-1 strain.