重组HIV-1壳体蛋白在转基因枸杞根系中的分泌表达

P24壳体蛋白（capsid, CA）是HIV¬-1早期感染的一个重要标志.用含有植物表达载体pCAMBIA 1305.2- MA4-CA（包含GRP信号肽和MA4-CA融合基因）的农杆菌菌株侵染枸杞,将转有MA4-CA融合基因的转化株诱导生根，并进行毛状根的培养; western blot证实根系及培养液中的MA4-CA融合蛋白以二聚体的形式存在，分子量为50 kDa；免疫组织化学显示，CA定位在细胞浆、细胞壁和细胞间隙中，充分证实了利用GRP信号肽可以引导重组蛋白分泌表达。建立枸杞中HIV-1壳体蛋白的根分泌表达系统，为研究植物HIV-1 CA-病毒样颗粒（VLPs）疫苗奠定基础。     

Retrovirus capsid protein assembly arrangements

During retrovirus particle assembly and morphogenesis, the retrovirus structural (Gag) proteins organize into two different arrangements: an immature form assembled by precursor Gag (PrGag) proteins; and a mature form, composed of proteins processed from PrGag. Central to both Gag protein arrangements is the capsid (CA) protein, a domain of PrGag, which is cleaved from the precursor to yield a mature Gag protein composed of an N-terminal domain (NTD), a flexible linker region, and a C-terminal domain (CTD). Because Gag interactions have proven difficult to examine in virions, a number of investigations have focused on the analysis of structures assembled in vitro. We have used electron microscope (EM) image reconstruction techniques to examine assembly products formed by two different CA variants of both human immunodeficiency virus type 1 (HIV-1) and the Moloney murine leukemia virus (M-MuLV). Interestingly, two types of hexameric protein arrangements were observed for each virus type. One organizational scheme featured hexamers composed of putative NTD dimer subunits, with sharing of subunits between neighbor hexamers. The second arrangement used apparent NTD monomers to coordinate hexamers, involved no subunit sharing, and employed putative CTD interactions to connect hexamers. Conversion between the two assembly forms may be achieved by making or breaking the proposed symmetric NTD dimer contacts in a process that appears to mimic viral morphogenesis.     

登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX—C和pLEX—Cv,转化E．coliGI724后用色氨酸诱导表达。经SDS—PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19％和13％。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。     

不同载体表达的人类细小病毒B19壳蛋白VP2的抗原性比较研究

构建表达质粒pcDNA3 VP2,将其转染CHO细胞建立了稳定表达的细胞系;用间接免疫荧光法和Western印迹证明了表达的VP2蛋白的特异性。对昆虫杆状病毒系统表达的VP2蛋白作初步纯化。分别用由大肠杆菌、CHO细胞和昆虫杆状病毒表达系统表达的VP2蛋白,以间接免疫荧光法和ELISA法检测人群血清中的VP2抗体,结果表明,间接免疫荧光法的敏感性高于ELISA法。     

戊型肝炎病毒IV型衣壳蛋白缺失突变体原核表达与性质

采用PCR技术扩增基因IV型HEV(Hepatitis E Virus,HEV)开放阅读框2(Open Reading Frame 2,ORF2)的缺失突变体(aa384-606),亚克隆到表达载体后,转化到大肠杆菌中进行诱导表达,表达蛋白命名为rP24。SDS-PAGE和免疫印迹实验表明,rP24获得了高效表达,且和单克隆抗体15B2具有强的反应活性。rP24经过包涵体洗涤、溶解复性、离子交换层析和分子筛层析纯化后,免疫印迹实验表明,纯化rP24能与抗HEV ORF2中和单克隆抗体8C11以及HE(Hepatitis E,HE)阳性血清发生很强的免疫反应性,说明rP24上具有构象型中和表位,模拟了HEV衣壳蛋白的空间结构。动态光散色测量结果表明,rP24的平均水化半径为7.48 nm;纯化rP24免疫动物实验表明,rP24具有强的抗原性,小鼠阳转周期短,抗体持续时间长;纯化rP24作为包被抗原检测HE阳性血清和阴性血清,结果显示rP24对HE阳性血清和阴性血清检出率与北京万泰公司的抗HEV-IgG检测试剂盒的检出率一致。这些实验结果说明,具有较好免疫反应性和免疫原性的rP24获得了高效表达,该蛋白模拟了天然病毒衣壳蛋白的中和表位,为进一步研究基因I型和基因IV型HEV感染不同宿主细胞差异的分子机制奠定了基础。     

人乳头瘤病毒16型L1蛋白的克隆及表达

采用PCR技术从宫颈癌组织中扩增人乳头瘤病毒16型(Human papillomavirus type16,HPV16)L1全长基因片段,目的片段克隆到pMD18T载体后经酶切鉴定及测序确认。构建重组原核表达质粒pGEX4T1-L1,转化大肠杆菌E.coliBL21,IPTG诱导表达出以非可溶性蛋白形式存在的表达蛋白,该重组蛋白的表达量占菌体总蛋白的17%,免疫印迹检测表明,表达蛋白与宫颈癌病人血清出现特异性反应。成功构建了重组原核表达质粒pGEX4T1-L1,并且在原核细胞中得到表达,为进一步研究L1蛋白的免疫学活性及疫苗的研发奠定了基础。     

HIV-1衣壳蛋白在转基因枸杞中表达的免疫组织化学定位

目的:研究转基因植物中重组蛋白的细胞定位,有助于进一步了解转基因枸杞中HIV-1衣壳(CA)蛋白融合蛋白的分泌表达途径。方法:利用含有MA4-CA融合基因的农杆菌转化枸杞,转化植株获得再生。采用免疫组织化学方法对转基因枸杞表达的CA融合蛋白进行初步定位。结果:免疫组织化学定位表明,在转基因枸杞愈伤组织中,HIV-1CA主要在细胞浆、细胞壁和细胞间隙中表达。结论:免疫组织化学结果初步证明了CA融合蛋白在转基因枸杞中的表达分布。     

3D reconstruction and capsid protein characterization of grass carp reovirus

Grass carp reovirus (GCRV) is a relatively new virus first isolated in China and is a member of the Aquareovirus genus of the Reoviridae family. Recent report of genomic sequencing showed that GCRV shared high degree of homology with mammalian reovirus (MRV). As a step of our effort to understand the structural basis of GCRV pathogenesis, we determined the three-dimensional (3D) structure of GCRV capsid at 17 Å resolution by electron cryomicroscopy. Each GCRV capsid has a multilayered organization, consisting of an RNAcore, an inner, middle and outer protein layer. The outer layer is made up of 200 trimers that are arranged on an incomplete T=13 icosahedral lattice. A characteristic feature of this layer is the depression resulting from the absence of trimers around the peripentonal positions, revealing the underlying trimers on the middle layer. There are 120 subunits in the inner layer arranged with T=1 symmetry. These structural features are common to other members of the Reoviridae. Moreover, SDS-PAGE analysis showed that GCRV virions contain seven structural proteins (VP1-VP7). These structural proteins have a high degree of sequence homology to MRV, consistent with the structural similarities observed in our study. The high structural similarities of isolated GCRV and MRV suggest that future structural studies focusing on GCRV entering into and replicating within its host cell are necessary in order to fully understand the structural basis of GCRV pathogenesis.     

猪圆环病毒3型衣壳蛋白(Cap)抑制宿主细胞天然免疫应答

【背景】猪圆环病毒是引起猪圆环病毒病的病原,能够引起严重的免疫抑制和临床症状,给养猪业造成严重的经济损失。【目的】研究猪圆环病毒3型(Porcinecircovirus3,PCV3)衣壳蛋白(Capsid protein,Cap)对宿主天然免疫应答的调控作用,解析其免疫抑制机制对阐明猪圆环病毒致病机制具有重要意义。【方法】通过构建Cap真核表达质粒,利用Westernblotting进行表达验证,实时荧光定量(RT-PCR)、双荧光素酶基因报告系统和ELISA探究Cap对I型干扰素通路活化的影响,通过免疫共沉淀探索其作用机制。【结果】真核表达质粒成功表达并且证实Cap可以抑制DNA模拟物Poly(dA:dT)诱导的I型干扰素通路的活化;Cap可以与天然免疫通路节点分子MITA相互作用。【结论】研究发现PCV3病毒Cap蛋白与干扰素通路节点分子MITA相互作用发挥免疫抑制作用,这为阐明PCV3免疫抑制机制提供了理论依据。     

3D reconstruction and capsid protein characterization of grass carp reovirus

Grass carp reovirus (GCRV) is the first aquatic vi-rus isolated and characterized in mainland China[1]. In 1983, it was reported that GCRV was the agent that caused severe outbreaks of infectious hemorrhage disease in grass carp (Cyenopharyngodon idellus). Subsequently, a series of relatively systematic analyses have been conducted to characterize the biological and molecular properties of GCRV[2-8]. More than 50 aquareoviruses have been identified since the first reovirus-like virus was…     

转基因枸杞中蛋白酶体糜蛋白酶样活性的研究

利用提取的转基因枸杞和正常枸杞蛋白与蛋白酶体的特异性荧光底物Suc-LLVY-AMC室温孵育,于360nm（激发光）/460nm（发射光）波长下测定荧光值的方法,研究了转基因枸杞和正常枸杞中蛋白酶体糜蛋白酶活性的差异及其特异性抑制剂MG115对枸杞中蛋白酶体活性的影响。结果表明表达H IV壳体蛋白的转基因枸杞蛋白酶体活性是空载体对照转基因枸杞蛋白酶体活性的3.6倍,是正常枸杞的4.2倍。10μM的MG115对表达H IV壳体蛋白的转基因枸杞蛋白酶体活性抑制率为87%,携带空载体对照的转基因枸杞抑制率为74%,而对正常枸杞抑制率仅为8.6%;50μM的MG115抑制作用与10μM的相比没有明显变化。这一结果将为利用蛋白酶体抑制剂提高转基因枸杞悬浮细胞H IV-1CA含量的研究提供理论依据。     

Conformational stability of dimeric and monomeric forms of the C-terminal domain of human immunodeficiency virus-1 capsid protein

The unfolding equilibrium of the C-terminal domain of human immunodeficiency virus-1 (HIV-1) capsid protein has been analyzed by circular dichroism and fluorescence spectroscopy. The results for the dimeric, natural domain are consistent with a three-state model (N(2)<-->2I<-->2U). The dimer (N(2)) dissociates and partially unfolds in a coupled cooperative process, into a monomeric intermediate (I) of very low conformational stability. This intermediate, which is the only significantly populated form at low (1 microM) protein concentrations, fully preserves the secondary structure but has lost part of the tertiary (intramonomer) interactions found in the dimer. In a second transition, the intermediate cooperatively unfolds into denatured monomer (U). The latter process is the equivalent of a two-state unfolding transition observed for a monomeric domain in which Trp184 at the dimer interface had been truncated to Ala. A highly conserved, disulfide-bonded cysteine, but not the disulfide bond itself, and three conserved residues within the major homology region of the retroviral capsid are important for the conformational stability of the monomer. All these residues are involved also in the association process, despite being located far away from the dimerization interface. It is proposed that dimerization of the C-terminal domain of the HIV-1 capsid protein involves induced-fit recognition, and the conformational reorganization also improves substantially the low intrinsic stability of each monomeric half.     

Cloning and expression of the HIV protein in <Emphasis Type="Italic">Escherichia coli</Emphasis> cell-free system

Sixteen kinds of human immunodeficiency virus (HIV) target genes were cloned by polymerase chain reaction (PCR) amplification, and specific plasmids were constructed as the templates for the expression of these genes in the cell-free system. Similarly, the linear PCR templates of these genes for cell-free protein expression were also constructed by using two PCR amplification process. These different templates can be employed to biosynthesize HIV proteins in the cell-free system simultaneously and can be adapted for some high-throughput processes. HIV protease (P10) was performed as a target protein, and two different templates (plasmid and PCR product) were prepared and used for P10 expression in the Escherichia coli cell-free system. The target protein P10 was detected in sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels either by using a plasmid template or by a PCR template. These results are promising and helpful to develop a high throughput process for drug discovery.     

鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体基因克隆构建及表达

为了获得原抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３＾１株轻链可变区基因,由连接肽体外连接获得单链抗体基因,在大肠杆菌中表达,从鼠源抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３株细胞中分离总ＲＮＡ,以ｏｌｉｇｏ（ｄＴ）１８为引物逆转录成ｃＤＮＡ,通过ＰＣＲ扩增出抗体的轻链（ＶＬ）和重链可变区（Ｖ１１）基因,由连接本外连接获得单链抗体（ＳｅＦｖ）基因。将此单链抗体（ＳｅＦｖ）基因插入原核表达载体ＰＥＴ２８ａ,经大肠杆菌（     

Production of HIV-1 p24 protein in transgenic tobacco plants

The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems. Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines. Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity. Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein. Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably. Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Novel epidithiodiketopiperazines as anti-viral zinc ejectors of the Feline Immunodeficiency Virus (FIV) nucleocapsid protein as a model for HIV infection

Focused libraries of multi-substituted epidithiodiketopiperazines (ETP) were prepared and evaluated for efficacy of inhibiting the nucleocapsid protein function of the Feline Immunodeficiency Virus (FIV) as a model for HIV. This activity was compared and contrasted to observed toxicity utilising an in-vitro cell culture approach. This resulted in the identification of several promising lead compounds with nanomolar potency in cells with low toxicity and a favorable therapeutic index.     

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI

Dengue virus (DV) is one of the most significanthuman viral pathogens transmitted by arthropod vectorsand now present in over 100 countries. Half of the world’spopulation live in areas at risk of dengue virus infection[1]. The infection of DV causes a spectrum of diseasesranging from a debilitating, self-limited illness (denguefever), and life-threaten syndromes (dengue haemorrhagicfever/dengue shock syndrome). Annually, the fourserotypes of DV collectively cause 50–100 million casesof inf…