H5N1 avian influenza virus HA protein of the pathogenic mechanism of immune injury

Title: H5N1 avian influenza virus HA protein of the pathogenic mechanism of immune injuryAuthor: Xu WeiDegree-granting unit: Guangzhou Medical CollegeKeywords: H5N1 avian influenza virus;; HA protein;; Insects - baculovirus expression system;; JAK-STAT signal transduction pathway;; NF-κB signaling
Transduction pathway;; SARS-CoVSummary:Objective:Human avian influenza (hereinafter referred to Magnetic lifter as bird flu) is a subtype of avian influenza virus in some strains of certain lead
From acute respiratory diseases, people infected with avian influenza virus (Avian influenza viruse, AIV) of mortality up to 60% or more of the
Significant human health threat. Recent studies suggest that high viral load in vivo and excessive immune inflammatory response caused by pathological damage
AIV is a major pathogenic mechanism. AIV through its surface membrane proteins - hemagglutinin (Hemagglutinin protein, HA) and target cells such as type II
Alveolar epithelial cells infected with the corresponding receptors into cells, a process that induces cytokines and chemokines, a large number of release into the
The activation and raise macrophages, neutrophils and http://www.999magnet.com/products/131-magnetic-lifter other inflammatory cells to the infection site, leading to severe diffuse alveolar damage, causing a
Similar to acute respiratory distress syndrome secondary to pathophysiological changes and the systemic multi-organ inflammatory immune response. So far, however, lack of
Lack of effects for the immune injury treatment. We are early in the SARS-CoV (severe acute respiratory syndrome
- Coronavirus) pathogenesis study found that: SARS-CoV Spike protein can activate the host innate immune response signaling pathways
, Induced cytokine / chemokine inflammatory cascade, and identified a key signaling molecule in target-JAK3 (Janus Kinase 3)
. As the AIV and SARS-CoV in the pathophysiology, clinical manifestations, and so has many similarities, we speculate that the avian influenza virus through
Through the surface film protein HA, may activate a common signaling molecules target the host innate immune-mediated pathological response, causing acute Free
Vaccine injury. The subject of the HA protein which attempts to reveal the damage caused by acute immune mechanisms of signal transduction molecules and key molecular target for exploration
AIV to discuss the invasion of immune and inflammatory response initiating, clarifying AIV and SARS-CoV may be a common molecular target, discovery and identification can be effective against
Virus-mediated immune damage lungs and other organs, the effective protection of the immune system drug targets, as lay the foundation for the development of innovative drugs.
Research contents and methods:1, the preparation of recombinant avian influenza virus (H5N1) HA proteinApplied Entomology - Table baculovirus eukaryotic
Of the system to express recombinant H5N1 HA protein, and by the nickel-affinity beads and Western blot identification of purified2, H5N1 HA protein
Induction of human lung adenocarcinoma epithelial cell line (A549) the possible mechanism of immune injuryDifferent concentrations of recombinant HA protein to stimulate the A549 cells for 1h, to mention
Take the total RNA; HA protein (40μg / ml) stimulated A549 cells at different times (15min, 30min, 1h, 2h, 4h) after extraction of total RNA, RT
-PCR detection of IRF-1 and IP-10 gene expression. Recombinant HA protein to stimulate A549 cells 12h, morphological changes were observed. Chip using liquid
Detection of supernatant cytokines and chemokines changes, observed changes in the dose-effect relationship. Examination by Western blot method
Measured JAK-STAT and NF-κB signal transduction pathway activation. Observation of JAK3 inhibitors on HA protein-induced IRF-1 and IP-10 gene expression
, Cytokines and chemokines change.3, the application of JAK3 knockout mice, targeting JAK3 for immune injury evaluation
The protective effectJAK3 knockout B6129S4-Jak3tm1Ljb mice (JAK3-/ -) and wild-type B6129SF2 / J mice
(JAK +/+), randomly divided into blank control group, PBS, HA protein group, by means of tracheal instillation into the HA protein, 72h after the separation of mouse lung and spleen
Dirty observed pathological changes.Results:1, the application of recombinant baculovirus system successfully expressed the H5N1 HA protein
Sequencing pFastbacHT-HA HA gene inserted plasmid from the A / chicken / Guangdong/191/04 (H5N1)
(GenBank; AY737289), homologous recombination is Bacmid-HA plasmid PCR products 4.1Kb, in line with expectations. Quality transfected Bacmid-HA
Tablets of sf9 cells, total protein was purified, Western blot identification confirmed as H5N1 avian influenza virus HA protein.2, H5N1 HA protein
Induction of human lung adenocarcinoma epithelial cell line (A549) the possible mechanism of immune injuryThe role of HA protein in A549 cells, the cell volume becomes large, thin
An increase in intracellular granules and vacuoles, the cells have rounded off, with time shows cell disruption solution; blank control group were not significantly different
Often. RT-PCR detected the HA protein-induced IRF-1 and IP-10 gene expression was significantly increased, a dose and time dependent; liquid microarray
The cell culture supernatant IL-6, IL-8, MCP-1, MIP-1alpha, MIP-1beta and RANTES expression was significantly increased and showed a significant amount of
Effect relationship. Western blot analysis found that the role of HA protein in A549 cells, the induction of STAT1, JAK2, JAK3, and phosphorylation of NF-κB
Of. Selective JAK3 inhibitors can down-regulate HA protein-induced IRF-1 and IP-10 gene expression, cytokine / inflammatory factors and suppressing the increase
System JAK3 and NF-κB phosphorylation.3, targeting JAK3 for HA-mediated immune injury has a protective effectJAK3 + / +
After tracheal infusion of HA protein in mice lung tissue pathology showed diffuse alveolar damage, and JAK3-/ - mice was significantly more lung injury
Light; JAK3 + / + mice spleen swelling, local structural damage germinal center, there are vacuolar changes, lymphopenia, and JAK3-/ - mice spleen
Was normal.Conclusion:A, H5N1 AIV HA protein by activating the JAK-STAT and NF-κB signal transduction pathway induced inflammation
Cascade.Two, JAK3 HA protein molecules are immune and inflammatory response triggered by the key early signaling molecules.Third, the election
Selective inhibition of JAK3 can induce immunity against the HA protein of the pathological damage, there will be potential drug targets; selective inhibition of JAK3
Agents may become the treatment of virus-related immune injury with promising agents.Degree Year: 2009