A型流感病毒血凝素蛋白S-棕榈酰化修饰的质谱分析

蛋白质S-棕榈酰化修饰是指棕榈酸分子通过硫酯键共价结合在蛋白质分子的半胱氨酸( S)的巯基侧链上,是蛋白质脂类修饰的重要形式之一,在细胞信号转导、代谢等过程中起着重要作用。本实验首先利用酰基-生物素置换反应,将A型流感病毒血凝素蛋白上的S-棕榈酸分子转换为含有生物素( Buotun)分子的标签。生物素标记蛋白经特异性富集、电泳分离纯化后,进行胶内水解。再利用质谱技术对水解混合物进行分析。结果表明,经酰基-生物素置换方法处理A型流感病毒裂解产物后,蛋白质耦联生物素的浓度(羟胺处理,＋Hydroxylamune)与空白对照组(未加羟胺处理,-Hydroxylamune)的比值大于3;对经富集后的流感病毒血凝素蛋白进行了质谱分析,鉴定了 A 型的两个 S-棕榈酰化修饰位点,分别位于蛋白羧基末端的 Cys562和Cys565。本研究为大规模研究S-棕榈酰化修饰蛋白提供了一种特异、有效的分析方法。     

Detection of influenza virus: traditional approaches and development of biosensors

Influenza is an acute respiratory disease caused by the influenza virus. The disease occurs annually, causing fatality in the elderly and children and billions of dollars loss in business and productivity. Traditional viral detection methods include MDCK cell culture, complement fixation, hemagglutinin-inhibition, and recently RT-PCR. Although effective, these methods generally involve labor-intensive laboratory procedures and often require trained personnel to carry them out. The development of biosensor technologies will enable rapid and specific disease diagnosis on-site so that a clinician can quickly determine whether treatment is needed. This paper reviews traditional viral assays and progress in the biosensor development for influenza virus. Recent advances in single-step direct detection using non-labeling techniques such as surface plasmon resonance, quartz-crystal microbalance, and colorimetric functional polymers are discussed.     

Testing of the influenza virus purification by CIEF

In virological practice, the pre‐concentration, purification and subsequent determination of the purity and concentration of the viruses from the cultural medium and/or from the real sample are required. The conventional techniques used today are equipment demanding, time‐consuming and laborious. In this study, the CIEF of influenza viruses with UV detection has been developed and subsequently used to test the purification of the virus from the biological samples. The equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs were precipitated by using PEG 6000 and sodium chloride. The precipitated viruses were centrifuged at 14 000×g, and the impurities of different densities were removed by using the sucrose gradients. The efficiency of the virus purification technique was examined by the CIEF and compared to the results of real‐time PCR. The pIs of both influenza viruses were determined. Simultaneously, the CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification.     

Pulmonary surfactant-derived antiviral actions at the respiratory surface

Lung surfactant (LS) is a membrane-based lipid-protein complex that lines the alveoli, reducing the surface tension at the air-liquid interface and thus minimizing the work of breathing. Besides this function, LS is also the first physical barrier between the outside air and the systemic circulation, therefore playing a key role in the defense against harmful particles and microorganisms.Viral respiratory tract infections (RTIs), and especially acute lower RTIs, are one of the leading causes of morbidity and mortality worldwide. LS participates in the network of interactions between viruses and the immune system to prevent or lessen the effects of the infection, but it is also altered by these pathogens, which can potentially impair its function.The aim of this review is to provide an integrated multidisciplinary overview toward understanding the interplay between respiratory viruses and LS and its health impact on the respiratory system. The review is centered on the antiviral mechanisms of both LS proteins and lipids, and their different interactions that lead to varying outcomes. Finally, a summary of the clinical application of surfactant in the scene of lung viral infection is disclosed, including state-of-the-art approaches of the therapeutic use of surfactant components.     

Apoferritin nanoparticle based dual-antigen influenza conjugate vaccine with potential cross-protective efficacy against heterosubtypic influenza virus

Ferritin nanoparticles with self-assembling properties have been widely explored as vaccine carrier by displaying foreign antigens through genetic fusion strategy. In the present work, an apoferritin (AFt) nanoparticle was tested as influenza vaccine carrier by chemically conjugating a matrix protein 2 ectodomain (M2e) antigen peptide or/and the full-length hemagglutinin (HA) antigen on the outer surface of the AFt, with heterobifunctional sSMCC or SM(PEG)24 containing PEG chain as linkers. To each AFt nanoparticle, about 30–32 M2e or 1.8 HA antigen could be coupled. The AFt-(PEG)₂₄-M2e, in which the M2e was coupled through SM(PEG)24 containing PEG chain, conferred higher protective efficacy in immunized mice than AFt-M2e did, but was less effective than AFt-(PEG)₂₄-HA. When both M2e and HA were coupled, the synthesized dual-antigen vaccine candidate AFt-(PEG)₂₄-M2e/HA elicited high level of M2e and HA antigen-specific antibodies and conferred 100% protection against lethal infection of homologous PR8 H1N1 virus strain and 70% protection against a heterologous A/FM/1/47 (FM1, H1N1) strain, which was more effective than the M2e or HA single antigen vaccine candidates. The potential cross-protective effect of the dual-antigen vaccine was further demonstrated by significant specific hemagglutination inhibition (HAI) titers in serum of the immunized mice against three other heterologous viral strains including A/Singapore/GP1908/2015 (IVR-180) H1N1, A/Anhui/1/2005 H5N1, and A/Hong Kong H3N2.     

糖链在流感病毒侵袭细胞中的作用

介绍了细胞外表面上含唾液酸的糖链在流感病毒侵袭细胞中的作用以及糖链与流感病毒表面糖蛋白－血凝素结合的特异性。     

Substituent effects on the binding of natural product anthocyanidin inhibitors to influenza neuraminidase with mass spectrometry

The binding of three closely related anthocyanins within the 430-cavity of influenza neuraminidase is studied using a combination of mass spectrometry and molecular docking. Despite their similar structures, which differ only in the number and position of the hydroxyl substituents on the phenyl group attached to the chromenylium ring, subtle differences in their binding characteristics are revealed by mass spectrometry and molecular docking that are in accord with their inhibitory properties by neuraminidase inhibition assays. The cyanidin and delphinidin, with the greatest number of hydroxyl groups, bind more strongly and are better inhibitors than pelargonidin that contains a lone hydroxyl group at the 4′ position. The study demonstrates, for the first time, the sensitivity of the mass spectrometry based approach for investigating the molecular basis and relative affinity of antiviral inhibitors, with subtly different structures, to their target protein. It has broader application for the screening of other protein interactions more generally with reasonable high-throughput.     

Electrochemical DNA biosensor based on avidin-biotin conjugation for influenza virus (type A) detection

An electrochemical DNA biosensor (E-DNA biosensor) was fabricated by avidin-biotin conjugation of a biotinylated probe DNA, 5′-biotin-ATG AGT CTT CTA ACC GAG GTC GAA-3′, and an avidin-modified glassy carbon electrode (GCE) to detect the influenza virus (type A). An avidin-modified GCE was prepared by the reaction of avidin and a carboxylic acid-modified GCE, which was synthesized by the electrochemical reduction of 4-carboxyphenyl diazonium salt. The current value of the E-DNA biosensor was evaluated after hybridization of the probe DNA and target DNA using cyclic voltammetry (CV). The current value decreased after the hybridization of the probe DNA and target DNA. The DNA that was used follows: complementary target DNA, 5′-TTC GAC CTC GGT TAG AAG ACT CAT-3′ and two-base mismatched DNA, 5′-TTC GAC AGC GGT TAT AAG ACT CAT-3′.