In Vivo Anti-inflammation Of Adenovirus Mediated Human TLR2 Gene

ObjectiveSepsis is a systemic inflammatory reaction syndrome (SIRS) caused by infection, which is a common complication of severe trauma, burn, shock and major operation, further development of sepsis could lead to septic shock and multiple organ dysfunction syndromes (MODS).Despite continuous development of antibiotics and improvement of procedures in recent years, mortality of sepsis remained high and became a tickler of intensive care. Previous research revealed that endotoxin (LPS) of gram-negative bacteria to be the major pathogen molecular of sepsis, which activated mononuclear phagocyte system, resulted to series of multiple inflammatory mediators, and the progression of disease. The newly developed and widely researched toll-like receptors (TLRs) was found to be highly related to immunoreactions by LPS and its consequent injuries, among which the relationship between toll-like receptor 2 (TLR2) and natural immunity has draw great attention.TLR2 was involved with human reaction to LPS and with the largest number of ligand recognition, which played a key role in triggering inherent immunity to different pathogenic microorganisms. It is confirmed that multiple inflammatory bacterial components could activate TLR2 directly and indirectly, which consequently activated leucocytes to eliminate bacteria, meanwhile different inflammatory mediums and cytokines were released, resulting inflammatory reactions; toxic shock syndromes (TSS) and complications might be induced by severe and extensive inflammation. Therefore, blocking or inhibition on the activation of TLR2 could effectively inhibit the activation of leucocytes, synthesis and secretion of inflammatory medium of leucocytes, and prevent severe inflammation caused by multiple bacterial components. Due to similar bacterial component affinity between soluble extracellular proteins of TLR2 (sTLR2) and natural TLR2 extracellular protein on leucocytes membrane, theoretically, sTLR2 could competitively inhibits the activation of corresponding receptor on leucocytes membrane, effective in anti-inflammation through blocking the activation of leucocytes and the release of inflammatory medium. Based on such theory, an adenovirus expression was selected in the study, human gene of TLR2 extracellular domain was cloned and analyzed by sequence homology comparison, a recombination of adenovirus vector with TLR2 extracellular domain was prepared, upon a high titer purified virus with sTLR2 expression was achieved; an animal model was created for the observation of anti-inflammatory effect of adenovirus mediated sTLR2, expected to provide a new method and agent of prevention and treatment of TSS and its complication, and speed up the research process.Methods1. Separation of peripheral blood mononuclear cell from healthy human was conducted; the RNA was extracted for the creation of a template for RT-PCR amplification of cDNA of TLR2 extracellular domain. Target gene was purified and sub-cloned to pUCm-T vector for the recombination of plasmid pUCm-TLR2, the accurate clones were selected by sequencing, and the results were compared for homology with human gene of TLR2 extracellular domain in GenBank.2. Correct target fragments were directional cloned to adenovirus shuttle plasmid pAdTrack-CMV for recombination of plasmid pAdTrack-CMV-TLR2, the positive recon was digested to linear and transferred to competent AdEasier-1 bacteria, and went through homogeneous recombination with adenovirus frame plasmid pAdEasy-1 in E.coli BJ5183 for the production of recombinant adenovirus plasmid pAd-TLR2.3. pAd-TLR2 were analyzed for recombination accuracy, and then packaged in 293 cell by liposome fusion and amplified. Expression of green fluorescence protein (GFP) were evaluated, the protein were purified and tested for virus titer, the recombinant adenovirus were analyzed by PCR and compared with wild-type virus for safety.4. A lethal dose of LPS induced inflammation animal model was established, the recombinant adenovirus AdTLR2 was applied pre and after inflammation, the impact on survival time was recorded.5. A small dosage LPS induced inflammation animal model was established too, and recombinant adenovirus AdTLR2 were injected through caudal vein pre and after inflammation, serum samples of different experiment groups were collected, alteration of cytokine TNF-α,IL-1β,IL-6,IL-10 and IL-13 were tested by ELISA, the influence of AdTLR2 on serum level of cytokines were evaluated. Results1. cDNA of TLR2 extracellular domain were amplified by RT-PCR from peripheral mononuclear cells of healthy human, and recombinant plasmid pUCm-TLR2 was successfully constructed.2. After sequencing and homologous comparison, segment size of target gene was confirmed as 1764 bp, the segment was free of base substitution and the same with TLR2 extracellular domain in GenBank.3. After double digestion and DNA sequencing, the insertion sequence and reading frame were proved to be correct, and the recombination of adenovirus shuttle plasmid pAdTrack-CMV-TLR2 was successful.4. Recombinant adenovirus plasmid pAd-TLR2 was created through homogeneous recombination of shuttle plasmid with adenovirus frame plasmid pAdEasy-1 in E.coli BJ5183, after PacⅠdigestion, a approximately 30 kb band and a 4.5 kb characteristic band was revealed, thus the homologous recombination was confirmed.5. Obvious green fluorescence was observed under fluorescent microscope, with a classical cytopathic effct (CPE) observed after the transportation of pAd-TLR2 to 293 cells, indicating successfully package of adenovirus.6. Recombinant adenovirus AdTLR2 with a titer of 3×109 PFU/mL was collected after cesium chloride density gradient centrifugation, the TLR2 extracellular domain was confirmed and free of wild-type virus by PCR.7. The endotoxemia inflammation animal model were established by intraperitoneal injection of BALB/c mouse, no significant toxicity of AdTLR2 on mouse were observed, the animal tolerated well on the virus.8. A prolonged survival time by AdTLR2 was observed on lethal dose of LPS animal model; serum level of TNF-α,IL-1β,IL-6,IL-10和IL-13 were reduced by AdTLR2 on small dosage of LPS animal model, indicating a certain immuno-regulation and anti-inflammatory effect of AdTLR2. Conclusion1. cDNA of TLR2 extracellular domain was successfully cloned, the target gene was free of base substitution and in accordance with corresponding series in GenBank, which was confirmed by sequencing and homologous comparison.2. Recombinant adenovirus plasmid AdTLR2 carriying TLR2 extracellular domain was successfully constructed by AdEasy System, upon amplication on 293 cell and purification, and recombinant adenovirus with a titer of 3×109 PFU/mL was produced.3. Recombinant adenovirus AdTLR2 resulted a prolong survival time of LPS induced inflammation animal model and reduction of serum level of TNF-α,IL-1β,IL-6,IL-10 and IL-13, which achieved a certain immuno-regulation and anti-inflammatory effect.