Preparation Of Monoclonal Antibody Against Eiger Of Musca Domestica And Study On Its Anti-stress Function

Musca domestica can survive and reproduce in the harsh environment enriched by various pathogenic microorganisms for its efficient innate immune system.The study of the disease resistance mechanism is greatly significant for the in-depth understanding of innate immunity of insects.The tumor necrosis factor(TNF)family members are important inflammatory cytokines in mammals,while those evolution is very conservative.Eiger,as an invertebrate TNF ortholog,was first discovered in Drosophila and has been confirmed to play a certain role in the immune response.Our laboratory previously reported a Eiger-like gene MdEiger in Musca domestica,which was significantly up-regulated after being stimulated by bacteria in housefly larvae.And MdEiger could cooperate with Toll and IMD signaling pathways to regulate the expression of antibacterial peptide genes and the activation of prophenoloxidase.Based on the above research work,this paper studies the expression changes of MdEiger under various stress conditions and its tissue localization.The results are as follows:1.The prokaryotic expression system was used to recombinantly express the TNF homology domain part of MdEiger.The recombinant product rMdEiger was used as immunogen to immunize mice.4 strains of anti-rMdEiger monoclonal antibodies were prepared and obtained by hybridoma technology.The purities,specificities,and titers of the antibodies all met the requirements of Western Blot and immunohistochemistry.2.Western Blot tests showed that two specific bands were detected on the cell membrane: mMdEiger1 and mMdEiger2.There were soluble MdEiger in the hemolymph of Musca domestica: sMdEiger1 and sMdEiger2.It would be the result of glycosylation modification of the digested product.And their contents would change with changes in the environment.3.Under the conditions of E.coli stimulation,UV irradiation,cadmium chloride and adriamycin exposures,the expression patterns of MdEiger were analyzed by qRT-PCR and Western Blot methods.It was found that MdEiger showed a significant up-regulation expression trend and the secretion and release of soluble protein MdEiger were stimulated under stress conditions.It is speculated that soluble MdEiger may play a pivotal role in regulating immune response,inflammatory response and oxidative stress.4.The expression localization analysis of MdEiger was performed by immunohistochemistry.Positive signals were detected in the fat cell membrane,the myometrium of the myocytes and many parts of the compound eyes.While it was mainly distributed in the cell membranes.5.The soluble recombinant protein r'MdEiger was successfully expressed using the Bac-to-Bac baculovirus expression system.The above research revealed the existence forms of Eiger in the housefly and its expression rules under various stress conditions.Based on this,it was speculated that Eiger mainly existed on the cell membranes of fat cells and nerve cells.When the housefly was subjected to environmental stress,the proteases on cell matrix or outer membrane would digest the extracellular part of Eiger,releasing a partial peptide containing TNF domain(sMdEiger)into the hemolymph.And as a cytokine regulated the stress response of Musca domestica by hemolymph circulation.This study could lay the foundation for the subsequent functional research of MdEiger and provided an important basis for revealing the molecular evolution and functional diversity of the TNF family.