Analysis Of Expression And Biological Function Of NK-Lysin, GM-CSF Based On Transcriptome Analysis At Sheep Alveolar Macrophage Induced By Lipopolysaccharide

Antibacterial peptide is a cysteine-rich, cationic peptides found in many biologies, which play an important role in innate immunity against bacteria, fungi, protozoa, and viruses. The bonds of the adaptive immue response and the acquired immune response.Antibacterial peptide fastly start the immune system, which has an important role in the body to resist pathogenic microbes. The alveolar macrophage is the main parts of the body to clearing pathogenic microorganisms. Lipopolysaccharide(LPS) is a component of gram-negative bacterial cell walls. LPS activates toll-like receptor(TLRs) to cause inflammation. Demonstrate the in vitro capacity of LPS to stimulate the secretion of proinflammatory cytokins through activation macrophage and monocyte cell. Therefore, we will find immune-related genes by Transcnptome sequencing technology base on alveolar macrophage in this study, and get gene to cloned、induced expression and tested biological function. It is provided an important basis for immune-enhancing adjuvant and breeding. The main tasks include as the following parts:(1)This study investigated the transcriptome profiles of alveolar macrophage using the Solexa RNA-Seq technologies. LPS-induced macrophage and uninduced macrophages were sequenced, looking for immune-related genes. To get67734Uningene by splicing of conting, which found54019new Unigene. However,64%sequence and bovine sequence were homologous.We discovered182of immune related genes by GO enrichment analysis of significantly differentially expressed genes. We found differentially expressed genes between Uninduced macrophages and induce macrophage, Looking for new gene in alveolar macrophages by using transcriptome and Solexa sequencing technology to feasible and effective. It was provide material for protein expression and biological function.(2)Expression of NK-Lysin in pichia pastoris. It clone a NK-Lysin cDNA of441bp. The NK-Lysin was insered into pichia pastoris constitutive expression vector. Recombinant plasmid pPIC9K-NK-Lysin identified by PCR, restriction analysis and sequencing, which was transformed into yeast GS115by electrotransformation. Postive bacteria was picked in4.0%Geneticn plates and inductioned by0.5%methanol, which was secretory expressed NK-Lysin. It was provided the basic for plenty sheep NK-Lysin.(3)Prokaryotic expression of NK-Lysin and protein activity tested. It is that successfully constructed a prokaryotic expression of NK-Lysin, and express fusion protein of37KDa induce in IPTG(0.5mmol/L) and37℃. Fusion protein of NK-Lysin was exhibited strong antibacterial abilities to E. coli and Listeria. It was provided a scientific basis for conducting antimicrobial peptide antibiotics substitute.(4)Induceding expression and tasting tissue distribution of NK-Lysin of sheep. To establish a specific fluorescence real-time quantitative method for detecting the expression of NK-Lysin mRNA in inducing conditions of concentration LPS and different immune tissues. The NK-Lysin mRNA expression in the differently induced conditions of LPS concentration, expression of4h was significant difference between the other groups(P<0.01)when LPS was0.1μg/mL, expression of8h reached a peak at0.01μg/mL LPS, NK-Lysin mRNA without expression in four time periods when LPS was100μg/mL. NK-Lysin mRNA have different levels of expression in shoulder lymph nodes, blood and splen, expression levels in the spleen was significant difference compared with the expression levels in other tissues(P<0.01). NK-Lysin gene expression levels related to the concentration and the induction time of LPS and having a specific distribution in the immune organs.(5)Prokaryotic expression of GM-CSF and protein activity tested. Cloning, expression and purification of sheep recombinant GM-CSF. The gene of GM-CSF was cloned into pET32α vector, and expressed in Escherichia coli strain BL21. The recombinant protein was purificated by AKTAxpressTM, MTT method was adopted to proliferative activity of GM-CSF which could stimulate proliferation of sheep peripheral blood iymphocytes. Its successful to clone a GM-CSF cDNA of435bp into pET32a vector, and express recombinant GM-CSF of35kDa induced in E.coli. Its purity highed than95%and concentration of860mg/mL. It has been identified by MTT that Fusion protein had an proliferative activity on cultivating lymphocytes of sheep, and the most efficacious concentration is200μg/mL. The consequences manifest that purity GM-CSF with good bioactivity was gained and provided an important basis for immune-enhancing adjuvant.The results above showed that library was constructes using transcriptome and Solexa sequencing technology to feasible and effective. We found182of immune related genes in library. The recombinant vector pPIC9K-NK-Lysin was constructed successfully and doing well expressed in yeast GS115; Recombination protein of PET32α-NK-Lysin exhibited strong antibacterial abilities to E. coli and Listeria; analysising the expression of NK-lysin gene by Q-RT-PCR. This will provide the basis for the selection disease resistance genes of breeding and immune-enhancing adjuvant.