Cloning And Expression Of MESK Gene Of Haemaphysalis Longicornis And Biological Characteristics

Tick is one of the most important pathogenic media, which spread various pathogenicmicroorganism including bacterium, virus, protozoon by bites and blood feeding on host epidermal. Theimmune system of host was induced by tick infestion, which caused host innate immunity and acquiredimmunity were actived. Immunomodulatory proteins derived tick regulate immune system of host whentick bited on host skin as well as involvement physiological and biochemical reactions and signalingpathways of host immune cells for persistent blood feeding. Tick-borne pathogenic microorganismsand non-Tick-borne pathogenic microorganisms were regulated simultaneously by tick. There are avariety of immune regulation proteins derived tick have been found, we exploit these proteins as targetsites of anti-tick vaccine, which was contributed to the research of theoretical basis and development ofcontrol ticks biological vaccine. Immunomodulatory protein derived tick was significance both inprevention and control tick-borne diseases. In addition, immunomodulatory protein also have a greatpotential to develop into drugs which functions is treated immune system diseases.MESK was amplified from a cDNA library of Haemaphysalis longicornis and was analysed bybioinfomatics, which was putative role that affect MAPK signaling pathway. MESK exact biologicalfunction have not been detailed exposition. The MESK3D crystal models of Haemaphysalislongicornis was structure by the swissmodel database service which utilized Ndrg2chain A crystalstructure of homo sapiens as template. Identities between the two crystal models was37.755%. BLASTresults revealed MESK and NDRG having high similarity, we uploaded MESK amino acid sequenceof Haemaphysalis longicornis to the conserved domain databasedatabase of NCBI and analysedsuperfamily relationship between MESK and NDRG, results showed that MESK and NDRG proteinfamily does not exist superfamily relationship, they just was multi-domains relationship. MESK andhelix-turn-helix protein family belonged to superfamily. Helix-turn-helix protein usual function wereone type trans-acting factor, which having two helical structure. The second helical identificate DNAand bind it, the first helical identificate other protein. The overall protein structure were composed oftwo helices and a corner. Therefore, we inferred MESK exert its physiological effect on cell by actingon the target mRNA or target protein. Scansite motifscan results revealed that MESK have a motif siteinteraction with14-3-3protein which played an important role on MAPK signal pathway. MESKprotein possessed four types of motif, which is tyrosine kinase site, threonine kinase site, DNA damagekinase and kinase binding site, therefore we inferred MESK have direct effect on MAPK signalingpathways. To understand the effect of MESK on mammalian cells and the influence of MESK tonon-tick-borne pathogenic microorganisms, we firstly structured the MESK of Haemaphysalislongicornis eukaryotic expression vector and transient transfection was successful in HeLa, HEK293and Vero cells. Experimental result showed MESK partly inhibited phosphorylation of ERK which isthe most classic MAPK in mammalian cell. ERK is the most important serine/threonine kinase inmammalian cells, which phosphorylated substrates were a considerable number of transcription factors, including c-fos, c-Jun, Elk-1, c-myc, ETS and so on. These transcription factors involved in cell growth,development, division, immune, cell function synchronization and other physiological reactions. MESKalso strongly promoted TTP expression at mRNA and protein level. TTP is one of trans-acting factors,which has been identified that specifically binds mRNAs containing Adenine/Uridine-Rich Elements(AREs) in3’-UTRs (3’-untranslatedregions) and directs them to exosome or P-body-mediateddegradation by reducing the target mRNA half-life, thus affecting the immune response of body. TTPinteracts with transcripts encoding for a number of cytokines such as TNF, IL-1β, IL-2, IL-6, GM-CSF,iNOS, COX-2and proteins(urokinase, urokinase receptor, VEGF). In addition, experiments resultsshowed that non-tick-borne pathogenic virus-avian influenza virus proliferation was inhibited due toMESK protien which inhibited phosphorylation of ERK in host cell. This suggested that MESK mayhave other available function.