Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray

Avian influenza virus (AIV) non-structural protein 1 (NS1) is a multifunctional protein. It is present at high levels in infected cells and can be used for AIV detection and diagnosis. In this study, we generated monoclonal antibody (MAb) D7 against AIV NS1 protein by immunization of BALB/c mice with purified recombinant NS1 protein expressed in Escherichia coli. Isotype determination revealed that the MAb was IgG₁/κ-type subclass. To identify the epitope of the MAb D7, the NS1 protein was truncated into a total of 225 15-mer peptides with 14 amino acid overlaps, which were spotted for a peptide microarray. The results revealed that the MAb D7 recognized the consensus DAPF motif. Furthermore, the AIV NS1 protein with the DAPF motif deletion was transiently expressed in 293T cells and failed to react with MAb D7. Subsequently, the DAPF motif was synthesized with an elongated GSGS linker at both the C- and N-termini. The MAb D7 reacted with the synthesized peptide both in enzyme-linked immunosorbent assay (ELISA) and dot-blot assays. From these results, we concluded that DAPF motif is the epitope of MAb D7. To our knowledge, this is the first report of a 4-mer epitope on the NS1 protein of AIV that can be recognized by MAb using a peptide microarray, which is able to simplify epitope identification, and that could serve as the basis for immune responses against avian influenza.     

A Hu sheep genome with the first ovine Y chromosome reveal introgression history after sheep domestication

The Y chromosome plays key roles in male fertility and reflects the evolutionary history of paternal lineages. Here, we present a de novo genome assembly of the Hu sheep with the first draft assembly of ovine Y chromosome(o MSY), using nanopore sequencing and Hi-C technologies. The o MSY that we generated spans 10.6 Mb from which 775 Y-SNPs were identified by applying a large panel of whole genome sequences from worldwide sheep and wild Iranian mouflons. Three major paternal lineages(HY1a, HY1b and HY2) were defined across domestic sheep, of which HY2 was newly detected. Surprisingly, HY2 forms a monophyletic clade with the Iranian mouflons and is highly divergent from both HY1a and HY1b. Demographic analysis of Y chromosomes, mitochondrial and nuclear genomes confirmed that HY2 and the maternal counterpart of lineage C represented a distinct wild mouflon population in Iran that diverge from the direct ancestor of domestic sheep, the wild mouflons in Southeastern Anatolia. Our results suggest that wild Iranian mouflons had introgressed into domestic sheep and thereby introduced this Iranian mouflon specific lineage carrying HY2 to both East Asian and Africa sheep populations.     

Variation of Two Primate Lineage-Specific Residues in Human SAMHD1 Confers Resistance to N Terminus-Targeted SIV Vpx Proteins

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) infection in myeloid cells but is inactivated by certain classes of simian immunodeficiency virus (SIV) Vpx proteins. Vpx proteins recruit the DCAF1-CRL4 E3 ubiquitin ligase to trigger species-specific SAMHD1 degradation. Determinants of SIV Vpx-mediated primate SAMHD1 degradation have been mapped to its C terminus. In this study, we have identified the N terminus of human SAMHD1 as a major species-specific determinant of Vpx-mediated suppression. The SIVmnd2 and SIVrcm Vpx proteins recognize the N terminus of rhesus, but not human, SAMHD1. We have also demonstrated that variation of two primate lineage-specific residues between human and rhesus SAMHD1 proteins determine resistance to SIVmnd2 and SIVrcm Vpx proteins. These residues (Cys15 and Ser52) are sequentially mutated to Phe in different lineages of Old World monkeys. Consequently, SIVmnd2 and SIVrcm Vpx proteins that could recognize Phe15- and Phe52-containing SAMHD1 could not inactivate human SAMHD1, which contains Cys15 and Ser52. In contrast, SIVmac Vpx, which targets the C terminus of SAMHD1 molecules, could inactivate various primate SAMHD1 molecules with divergent C-terminal sequences. Both C terminus-targeted SIVmac Vpx and N terminus-targeted SIVrcm Vpx require DCAF1 for the induction of SAMHD1 degradation. The ability of SIV Vpx to restrict SAMHD1 among different primate species is a manifestation of the SAMHD1 evolutionary pattern among those species.     

利用废弃花材简易制作压花画

利用花店、苗圃、公园、花农等废弃的大量可利用的花材,以及大自然中的小草野花进行简易的选材、收集、整理、干燥与保存,经过艺术加工后,制作出具有较强观赏性的压花画,并对压花画的保存进行了介绍。     

α-氨基酸酯酰基转移酶表达纯化、酶学性质及其催化应用

α-氨基酸酯酰基转移酶(α-amino acid ester acyltransferase,AET)能够催化底物L-丙氨酸甲酯盐酸盐、L-谷氨酰胺合成L-丙氨酰-L-谷氨酰胺(L-alanyl-L-glutamine,丙谷二肽)。利用重组大肠杆菌saet-QC01表达α-氨基酸酯酰基转移酶,对其表达条件进行了优化,通过Ni-NTA亲和层析法分离纯化重组蛋白,并对其酶学性质、催化应用进行了研究。适合酶表达的诱导条件:温度20℃,诱导阶段(OD_(600)=2.0-2.5),IPTG浓度0.6 mmol/L,诱导时间12 h。α-氨基酸酯酰基转移酶的最适反应温度27℃,最适pH 8.5,在pH 7.0-8.0很稳定,在酸性条件下相对稳定,低浓度的Co~(2+)、低浓度的EDTA对酶活有促进作用。在底物浓度丙氨酸甲酯盐酸盐600 mmol/L、谷氨酰胺480 mmol/L,丙谷二肽的产量达到78.2 g/L,生产速率达到1.955 g/(L·min),转化率达到75.0%。α-氨基酸酯酰基转移酶具有良好的酸碱耐受性,催化效率高的优良特性,在工业生产中具有较好的应用潜力。     

抗多药耐药靶点及相关药物的研究进展

抗多药耐药是指在疾病的治疗过程中,细胞对多种药物产生广泛的耐受,导致治疗效果不理想的现象。多药耐药多发于感染与肿瘤疾病的治疗中,已成为治愈这2 类疾病的主要障碍。从转运蛋白和离子通道、酶以及核糖体这3 个方面综述抗多药耐药靶点的机制及相关药物的研究进展,旨在为抗多药耐药药物的研发提供参考。     

Molecular Insight into the Steric Shielding Effect of PEG on the Conjugated Staphylokinase: Biochemical Characterization and Molecular Dynamics Simulation

PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.