鸭源H9N2亚型禽流感病毒血凝素基因的克隆、序列分析及原核表达

参照GenBank收录的H9N2亚型禽流感病毒血凝素(HA)基因序列设计并合成引物,以鸭源H9N2亚型禽流感病毒RNA为模板,利用RT PCR方法扩增了预计约1700bp的HA基因,将此扩增产物克隆进pMD18 T载体,采用限制性酶切及序列测定鉴定阳性重组克隆子.测序结果表明HA基因长为1683bp.基于HA蛋白的信号肽在表达中的负面作用,本研究通过基因工程手段缺失HA蛋白位于起始的信号肽的编码序列,获得了缺失HA蛋白信号肽的HA基因,并将其亚克隆到pGEX KG中,与GST融合表达.SDS PAGE结果显示:融合表达的蛋白分子量约为90×103.Western印迹表明表达蛋白具有免疫活性.     

Molecular basis of the structure and function of H1 hemagglutinin of influenza virus

Influenza virus hemagglutinin (HA) contains antigenic sites recognized by the host immune system, cleavage sites cleaved by host proteases, receptor binding sites attaching to sialyl receptors on the target cell, and fusion peptides mediating membrane fusion. Change in an amino acid(s) in these sites may affect the potential of virus infection and spread within and between hosts. Influenza viruses with H1 HA infect birds, pigs and humans and have caused two of the four pandemics in the past 100 years: 1918 pandemic that killed 21-50 million people and 2009 pandemic that caused more than 18,000 deaths. Understanding the relationship between antigenic structure and immune specificity, the receptor binding specificity in virus transmission, how the cleavage site controls pathogenicity, and how the fusion peptide causes membrane fusion for the entry of influenza virus into the host cell should provide information to find more effective ways to prevent and control influenza.     

Intermolecular Mechanism and Dynamic Investigation of Avian Influenza H7N9 Virus’ Susceptibility to E119V-Substituted Peramivir–Neuraminidase Complex

The H7N9 virus attaches itself to the human cell receptor protein containing the polysaccharide that terminates with sialic acid. The mutation of neuraminidase at residue E119 has been explored experimentally. However, there is no adequate information on the substitution with E119V in peramivir at the intermolecular level. Therefore, a good knowledge of the interatomic interactions is a prerequisite in understanding its transmission mode and subsequent effective inhibitions of the sialic acid receptor cleavage by neuraminidase. Herein, we investigated the mechanism and dynamism on the susceptibility of the E119V mutation on the peramivir–neuraminidase complex relative to the wildtype complex at the intermolecular level. This study aims to investigate the impact of the 119V substitution on the neuraminidase–peramivir complex and unveil the residues responsible for the complex conformations. We employed molecular dynamic (MD) simulations and extensive post-MD analyses in the study. These extensive computational investigations were carried out on the wildtype and the E119V mutant complex of the protein for holistic insights in unveiling the effects of this mutation on the binding affinity and the conformational terrain of peramivir–neuraminidase E119V mutation. The calculated total binding energy (ΔG_bind) for the peramivir wildtype is −49.09 ± 0.13 kcal/mol, while the E119V mutant is −58.55 ± 0.15 kcal/mol. The increase in binding energy (9.46 kcal/mol) is consistent with other post-MD analyses results, confirming that E119V substitution confers a higher degree of stability on the protein complex. This study promises to proffer contributory insight and additional knowledge that would enhance future drug designs and help in the fight targeted at controlling the avian influenza H7N9 virus. Therefore, we suggest that experimentalists collaborate with computational chemists for all investigations of this topic, as we have done in our previous studies.     

On-chip graphene oxide aptasensor for multiple protein detection

The versatility of an on-chip graphene oxide (GO) aptasensor was successfully confirmed by the detection of three different proteins, namely, thrombin (TB), prostate specific antigen (PSA), and hemagglutinin (HA), simply by changing the aptamers but with the sensor composition remaining the same. The results indicate that both DNA and RNA aptamers immobilized on the GO surface are sufficiently active to realize an on-chip aptasensor. Molecular selectivity and concentration dependence were investigated in relation to TB and PSA detection by using a dual, triple, and quintuple microchannel configuration. The multiple target detection of TB and PSA on a single chip was also demonstrated by using a 2 × 3 linear-array GO aptasensor. This work enables us to apply this sensor to the development of a multicomponent analysis system for a wide variety of targets by choosing appropriate aptamers.     

Cover Picture: Gold Nanoparticles Decorated with Sialic Acid Terminated Bi‐antennary N‐Glycans for the Detection of Influenza Virus at Nanomolar Concentrations (ChemistryOpen 6/2015)

Gold nanoparticles decorated with full‐length sialic acid terminated complex bi‐antennary N‐glycans, synthesized with glycans isolated from egg yolk, were used as a sensor for the detection of both recombinant hemagglutinin (HA) and whole influenza A virus particles of the H1N1 subtype. Nanoparticle aggregation was induced by interaction between the sialic acid termini of the glycans attached to gold and the multivalent sialic acid binding sites of HA. Both dynamic light scattering (DLS) and UV/Vis spectroscopy demonstrated the efficiency of the sensor, which could detect viral HA at nanomolar concentrations and revealed a linear relationship between the extent of nanoparticle aggregation and the concentration of HA. UV/Vis studies also showed that these nanoparticles can selectively detect an influenza A virus strain that preferentially binds sialic acid terminated glycans with α(2→6) linkages over a strain that prefers glycans with terminal α(2→3)‐linked sialic acids.     

Theoretical investigation on the binding specificity of fluorinated sialyldisaccharides Neu5Acα(2–3)Gal and Neu5Acα(2–6)Gal with influenza hemagglutinin H1 – A Molecular Dynamics Study

In the present study, the hydroxyl groups at the C4 and C7 positions of sialic acid and C6 position of galactose in Neu5Acα(2–3)Gal (N23G) and the hydroxyl groups at the C8 position of sialic acid and C3 and C4 positions of galactose in Neu5Acα(2–6)Gal (N26G) were substituted with fluorine atoms, respectively. Molecular dynamics simulations of 100 ns duration were carried out to investigate the structural and dynamical behavior of H1 bound with the tri-fluorinated N23G and N26G (FN23G and FN26G). Based on energy analysis, it was concluded that FN26G should be a better binder for hemagglutinin (H1) than FN23G and it might act as an inhibitor for influenza.     

Label‐Free Detection of Glycan–Protein Interactions for Array Development by Surface‐Enhanced Raman Spectroscopy (SERS)

A glyco‐array platform has been developed, in which glycans are attached to plasmonic nanoparticles through strain‐promoted azide‐alkyne cycloaddition. Glycan–protein binding events can then be detected in a label‐free manner employing surface‐enhanced Raman spectroscopy (SERS). As proof of concept, we have analyzed the binding of Gal1, Gal3, and influenza hemagglutinins (HAs) to various glycans and demonstrated that binding partners can be identified with high confidence. The attraction of SERS for optical sensing is that it can provide unique spectral signatures for glycan–protein complexes, confirm identity through statistical validation, and minimizes false positive results common to indirect methods. Furthermore, SERS is very sensitive and has multiplexing capabilities thereby allowing the simultaneous detection of multiple analytes.     

Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV–N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV–N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV–N, bound to HA with a maximum K_D of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar K_D, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV–N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.     

Synthesis of Four Pentacyclic Triterpene–Sialylglycopeptide Conjugates and Their Affinity Assays with Hemagglutinin

Influenza outbreaks pose a serious threat to human health. Hemagglutinin (HA) is an important target for influenza virus entry inhibitors. In this study, we synthesized four pentacyclic triterpene conjugates with a sialylglycopeptide scaffold through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) and prepared affinity assays of these conjugates with two HAs, namely H1N1 (A/WSN/1933) and H5N1 (A/Hong Kong/483/97), respectively. With a dissociation constant (K_D) of 6.89 μM, SCT-Asn-betulinic acid exhibited the strongest affinity with the H1N1 protein. Furthermore, with a K_D value of 9.10 μM, SCT-Asn-oleanolic acid exhibited the strongest affinity with the H5N1 protein. The conjugates considerably enhanced antiviral activity, which indicates that pentacyclic triterpenes can be used as a ligand to improve the anti-influenza ability of the sialylglycopeptide molecule by acting on the HA protein.