Construction Of A CDNA Librarie From LPS Stimulus Induced Ovine Primary Alveolar Macrophages, EST Analysis And Study On Several Immune-related Genes

Lipopolysaccharide (LPS) is a predominant glycolipid of the outer membrane of gram-negative bacteria and plays a key role in gram-negative bacteria infectious disease.It can stimulate monocytes, macrophages, and neutrophils to produce cytokines; increases the expression of cell-adhesion molecules and induces the secretion of proinflammatory mediators.The aim of the study was to construct full-length cDNA library from LPS induced ovine alveolar macrophage and clone some of immune-related genes such as cytokines,toll-like receptors and defensin,detect the dynamics of these genes expression during LPS stimulation, clarify the molecular mechanism of cytokines,toll-like receptors(TLRs)and defensin participating in immune response. Through genetic engineering to express immune-related genes and identify its biological function, lay the foundation of the development for immune adjuvant and therapeutic drugs.At present, The number of sheep cDNA library in various cell types is seldom, and provide very limited genetic markers for the sheep genomic research, resulting in physical map of sheep genome is lagging behind than other domestic animals such as cattle, pigs. Sheep genomic function studies focused on traits of economic-related genes, however ,The immune-related factors research are still far from full. The mechanism of regulation on sheep cytokines(interleukin [IL]-1β, IL-8, granulocyte-macrophage colony stimulating factor [GM-CSF], and interferon [IFN]-γ), toll-like receptors and defensin is still not clear, but it is of great significance for elucidating pathologic process and is necessary to study in-depth.Ovine primary alveolar macrophages were cultured in vitro and identified with trypan blue exclusion and phagocytic experiment, The SMART technology was used to construct a full-length cDNA library from LPS induced ovine alveolar macrophages. Random of 255 clones of cDNA library were sequenced and align with BLAST/n and BLAST/x,the new expressed sequence tags(ESTs) were annotated and submitted to GenBank. Some of immue-related genes such as cytokines(IL-1β,IL-8,GM-CSF,IFN-γ),TLRs ,defensin and housekeeping genes(G3PDH,α-Tubulin)were cloned and sequenced with molecular biologic methods.The mRNA level of these genes response to LPS stimulated alveolar macrophage were investigated by using real-time RT-PCR. The distribution and variance of sheep beta-defensin1(sBD1) in developmental stages was determined by RT-PCR and real-time PCR.The response of sBD1 and IL-8 in Salmonella infective models were detected by real-time PCR.The recombinant sheep GM-CSF expression vector were constructed and transferred into yeast by electric shocks, and expressed secretroyly with methanol induction. Sheep beta-defensin1 were expressed in fusion in E.coli.Preliminary results as following: (1) cDNA library from LPS induced sheep alveolar macrophage was constructed, the capacity was 1.6×106 pfu/ml and recombinant rate reached 95.7%.45 new ESTs or genes from 255 effective EST sequences were submit to GenBank,the accession numbers were EU366470-EU366473, EU366475-EU366492, EU366494-EU366497,EU370534-EU370552, and discovered 6 unknown EST sequences.(2) The regular pattern of several cytokines(IL-1β,IL-8,GM-CSF,IFN-γ) and TLR family response to LPS stimuli were elucidated systematically. The mRNA expression of IL-1β,IL-8,GM-CSF were peaked between 4 and 12 h, the exception was IFN-γmRNA whose expression peaked around 16 h post-stimulation.The expression of TLR1,2,4,6,7,8,9,10 were detected exception that of TLR3,5.Further, TLR2 ,4 mRNA expression rapidly increased post-stimulation and peaked at 20 min post-stimulation and this high level was maintained throughout the procedure.(3)sheep alveolar macrophage was not target cell for sBD1 expression ,It was expressed consistently in the tissue of digestive and respiratory tract, the expression level in digestive tract was neonatal> adult>fetal sheep. The decreased change in expression of sBD1 was detected in digestive tract during Salmonella infection while IL-8 was Significant upregulation under this conditions.(4) Sheep GM-CSF was secreted expression in picha yeast and accouted for 17% of total protein; sBD1 were expressed in E.coli, But did not show antibacterial activity.The above results showed that we have firstly constructed a cDNA library of sheep alveolar macrophage induced with LPS,it provide a platform for the function of immune-related genes,the new EST accoutting for 20% enriched the genetic markers of sheep genome physical map;The regular pattern of the cytokines(IL-1β,IL-8,GM-CSF,IFN-γ) offer the scientific basis for elucidating the molecular mechanism of pathogenesis.Alveolar macrophage is not target cell for the expression of TLR3,5 and sBD1 while TLR2,4 are sensitive for LPS stimulation ,TLR2,4 plays an important role in response to gram-negative bacteria infection;The expression of sBD1 exclusively and consistently in mucous system show that it plays an important role in innate immunity,there was no response to the samonella infection may be due to its not directly involved in the resistance of bacterial infection but related with other type of pathogens.The IL-8 significantly participated in Anti-infective immunity for salmonella infection;Sheep GM-CSF involved in the early stage immune response,and shown a good prospect for development of adjuvant vaccine and therapeutic drug, so we expressed sheep GM-CSF in yeast for the preparation of its development and application; sBD1 in mucous system widely expressed but its biological function is unclear, sheep defensin-1 expressed in E.coli offere the opportunity to research its biologic function in vitro.