Baculoviral capsid display of His-tagged ZnO inorganic binding peptide

Virus-templated fabrication of compound structures can be made through incorporating the specifically inorganic-binding peptide into the viral scaffold, widely used is phage display system. Compared to prokaryotic phages, insect cell-based baculovirus has some strengths such as the adaptability to the proteins’ posttranslational modification and non-replication in mammalian cells. As an attempt to explore the baculovirus-mediated bioconjugates, we show in this study that a genetically engineered baculovirus, with a hexahistidine (His₆) tagged ZnO binding peptide fused to the N-terminus of the viral capsid protein vp39 of AcNPV, was constructed. It maintains both the viral infectivity and the fusion protein’s activity. The presence of the fusion protein on the baculovirus particle was demonstrated by western blot analysis of purified budded virus. Its display on the virus capsid was revealed by virus fractionation analysis. The binding of nanosized ZnO powders to the virus capsid was visualized by transmission electron microscopy (TEM). This is the first report of the display of the inorganic-binding peptide on the capsid of eukaryotic baculovirus. Aimed at the nanomaterials’ application in the biological field, this research could find useful in the biotracking of the baculovirus transduction process and the preparation of novel functional nanodevices.     

Time-resolved molecular dynamics of bacteriophage HK97 capsid maturation interpreted by electron cryo-microscopy and X-ray crystallography

The bacteriophage HK97 capsid is a molecular machine that exhibits large-scale conformational rearrangements of its 420 identical protein subunits during capsid maturation. Immature empty capsids, termed Prohead II, assemble in vivo in an Escherichia coli expression system. Maturation of these particles may be induced in vitro, converting them into Head II capsids that are indistinguishable in conformation from the capsid of an infectious phage particle. One method of in vitro maturation requires acidification to drive the reaction through two expansion intermediates (EI-I, EI-II) to its penultimate particle state (EI-III), which has 86% more internal volume than Prohead II. Neutralization of EI-III produces the fully mature capsid, Head II. The three expansion intermediates and the acid expansion pathway were characterized by cryo-EM analysis and 3D reconstruction. We now report that, although large-scale structural changes are involved, the electron density maps for these intermediate states are readily interpreted in terms of quasi-atomic models based on subunit structures determined by prior crystallographic analysis of Head II. Progression through the expansion intermediate states primarily represents rigid-body rotations and translations of the subunits, accompanied by refolding of two small regions, the N-terminal arm and a beta-hairpin called the E-loop. Movies made with these pseudo-atomic coordinates and the Head II X-ray coordinates illuminate various aspects of the maturation pathway in the course of which the pattern of inter-subunit interactions is sequentially transformed while the integrity of the capsid is maintained.     

Multiple Roles of HIV-1 Capsid during the Virus Replication Cycle

Human immunodeficiency virus-1 capsid(HIV-1 CA) is involved in different stages of the viral replication cycle. During virion assembly, CA drives the formation of the hexameric lattice in immature viral particles, while in mature virions CA monomers assemble in cone-shaped cores surrounding the viral RNA genome and associated proteins. In addition to its functions in late stages of the viral replication cycle, CA plays key roles in a number of processes during early phases of HIV-1 infection including trafficking, uncoating, recognition by host cellular proteins and nuclear import of the viral preintegration complex. As a result of efficient cooperation of CA with other viral and cellular proteins, integration of the viral genetic material into the host genome, which is an essential step for productive viral infection, successfully occurs. In this review, we will summarize available data on CA functions in HIV-1 replication, describing in detail its roles in late and early phases of the viral replication cycle.     

Prokaryotic Expression and Potential Application of the Truncated PCV-2 Capsid Protein

Three pairs of specific primers were designed to amplify the F2-1, F2-2 and XF2-2 truncated sequences of ORF2 which encodes the capsid protein of porcine circovirus type 2 (PCV-2). The F2-1 sequence had most of the NLS region of ORF2, but the F2-2 and XF2-2 genes had the NLS region deleted. Truncated genes were subcloned into pET-32a(+) vectors to construct recombinant fusion expression vectors. The vectors were then transformed into Rosetta(DE3) E. coli and expressed by induction of IPTG. Expressed protein...     

豇豆花叶病毒衣壳蛋白编码区在中间组份的RNA核苷酸序列上的定位(英文)

对豇豆花叶病毒两个衣壳蛋白(VP37和VP23)的氨基端和羧基端氨基酸序列进行了分析,这些结果可以允许VP37和VP23编码区在病毒中间组份(M)RNA的核苷酸序列上进行基因定位。这两个编码区是相邻的,并表明,从M RNA的原始翻译产物中释出VP37和VP23的蛋白酶解部位,分别是谷氨酰胺-甲硫氨酸和谷氨酰胺-甘氨酸二肽序列。     

Protective immunity conferred by porcine circovirus 2 ORF2‐based DNA vaccine in mice

Post‐weaning multisystemic wasting syndrome (PMWS) associated with porcine circovirus type 2 (PCV2) has caused the swine industry significant health challenges and economic damage. Although inactivated and subunit vaccines against PMWS have been used widely, so far no DNA vaccine is available. In this study, with the aim of exploring a new route for developing a vaccine against PCV2, the immunogenicity of a DNA vaccine was evaluated in mice. The pEGFP‐N1 vector was used to construct a PCV2 Cap gene recombinant vaccine. To assess the immunogenicity of pEGFP‐Cap, 80 BALB/c mice were immunized three times at 2 weekly intervals with pEGFP‐Cap, LG‐strain vaccine, pEGFP‐N1 vector or PBS and then challenged with PCV2. IgG and cytokines were assessed by indirect ELISA and ELISA, respectively. Specimens stained with hematoxylin and eosin (HE) and immunohistochemistry (IHC) techniques were examined histopathologically. It was found that vaccination of the mice with the pEGFP‐Cap induced solid protection against PCV2 infection through induction of highly specific serum IgG antibodies and cytokines (IFN‐γ and IL‐10), and a small PCV2 viral load. The mice treated with the pEGFP‐Cap and LG‐strain developed no histopathologically detectable lesions (HE stain) and IHC techniques revealed only a few positive cells. Thus, this study demonstrated that recombinant pEGFP‐Cap substantially alleviates PCV2 infection in mice and provides evidence that a DNA vaccine could be an alternative to PCV2 vaccines against PMWS.     

鸡贫血病毒衣壳蛋白的表达纯化及多克隆抗体制备

利用PCR技术,以pPrpo-VP1为模板扩增得到鸡贫血病毒的衣壳蛋白基因(VP1),以T4多聚核苷酸激酶磷酸化处理、纯化后,克隆至表达载体pET-30a(+) 中,从而构建了原核表达质粒pET30-VP1.将pET30-VP1转化至感受态细胞E.coli BL21(DE3)中,经IPTG诱导后,SDS-PAGE分析,可见约45kDa的目的蛋白获得表达.该蛋白经亲和层析纯化后,免疫6-8w的雌性Balb/c鼠,三次免疫后,采血分离血清,制得抗VP1的多克隆血清.以纯化的VP1为包被抗原,用ELISA方法检测,制备的血清效价达12800×以上.以Western blot 检测,该血清可与目的蛋白发生特异性反应,证明其具有良好的免疫原性.VP1蛋白的成功表达及其多克隆抗体的制备为进一步研究VP1蛋白的功能及开展CAV疫苗及诊断制剂的研制奠定了基础.     

来源不同V-C组不同的小麦全蚀病菌病毒的特性

从地理位置不同,V—C组不同的13株小麦全蚀病菌中分离到直径23—36纳米的病毒。聚丙烯酰胺凝脉电泳表明这些菌株中的病毒dsRNA的组分及分子量不同。泰安株病毒有分子量为3.45,2.95,1.60,1.52,1.30,1.25,1.15,1.00,<1.00×10~6的9个dsRNA,而浙江、湖北菌株仅有一个组分、分子量为3×10~6。多数病毒衣壳多肽的的分子量为66×10~3—73×10~3。泰安菌株与烟台菌株病毒抗血清除与张家口菌株、青海菌株无反应外与其它菌株病毒均有反应,但在免疫双扩散中抗血清的滴定度不同。