Study On Biologicla Function Of SPLUNC1 Protein Of Xinjiang Bashibai Sheep

Short palate, lung and nasal epithelium clone 1( SPLUNC1) is highly expressed specifically in the respiratory tract which is a secreted protein involved in immune defenses, with the function of anti-infection, cleared harmful microorganisms, tumor suppressor, anti-tumor. SPLUNC1 as a natural protective immune proteins, the maintenance of the upper respiratory tract of normal physiological activity plays an important role. But for SPLUNC1 research concentrated on the human and mouse, no research reports on Bashibai sheep.Objective: In this study, based on the successful expression of recombinant protein Bashibai sheep SPLUNC1(r SPLUNC1), purification and western blot detection, and further research into r SPLUNC1 biological functions in vitro, including Mycoplasma ovipneumoniae(Mo) value-added impact on pathogens antibacterial activity, regulation of the activity of neutrophil elastase(NE), and bacterial infections of the outer peripheral lymphocyte apoptosis. It is laid the foundation to further study the biological function of r SPLUNC1.Methods:(1)Purification and western blot detection of r SPLUNC1: On the basis of the constructed Bashibai sheep recombinant Pichia GS115/p PIC9K-SPLUNC1 negative and positive clones clone on methanol-induced expression of 96 h supernatant was collected, using Ni-NTA agarose affinity chromatography, and Western Blot method to detect the effect of purification.(2)Using plate count and real-time PCR, two ways to detect the effect of three different concentrations r SPLUNC1(10μg/m L, 20μg/m L, 40μg/m L) on the growth of Mo.Using stereoscopic microscope to observe single colony count;using fluorescence quantitative PCR detection of Mo 16 S r RNA copy number.(3)Lung pathogens antibacterial activity of r SPLUNC1: using microdilution to determine different solubility r SPLUNC1(40μg/ml, 20μg/ml, 10μg/ml, 5μg/ml) for Pasteurella, Escherichia coli, Streptococcus pneumoniae, Staphylococcus antibacterial activity of this four kinds of staphylococcus bacteria.Cultured various concentrations r SPLUNC1 with test bacteria seted 96-well, measured OD600 nm at 0h, 1h, 2h, 3h, 4h, 5h, 6h.(4)Regulation r SPLUNC1 to NE: Regulation of chromogenic substrate assay r SPLUNC1(40μg/ml, 20μg/ml, 10μg/ml) of NE activity.added Mo stimulated neutrophils, 2h after the supernatant was collected,transferred to a 96 well plate with 0.1M HEPES mixed,added 2 mmol/L tetrapeptide substrate, 37 ℃ incubated for 5min then determined OD405 nm value.(5)r SPLUNC1 peripheral lymphocyte apoptosis: The lymphocytes were infected with Mo, Pasteurella, Streptococcus pneumoniae, three kinds of respiratory pathogens, then added r SPLUNC1 protein, lymphocyte apoptosis detected by fluorescence staining further by agarose gel electrophoresis to detect apoptosis DNA Ladder.Results and conclusions:(1) r SPLUNC1 Protein purification, the hybrid protein significantly reduced, SDS-PAGE analysis shows protein molecular size 25.53 k Da purposes strip; Western Blot can be detected with an anti-His-tagged murine monoclonal antibody that specifically binds to the target band, protein single stripe, the size of 25.53 Kda, consistent with the expected results. Description successfully obtained r SPLUNC1 protein, provide the basis for subsequent experiments.(2) Plate count in, 10 μg/m L, 20 μg/m L, 40 μg/m L r SPLUNC1 concentrations colonies were reduced by 37.25%(p<0.01) than the control group, 48.74%(p<0.01), 67.23%(p< 0.01); fluorescence quantitative PCR, and from 2 h onwards, the experimental group Mo 16 S r RNA copy number decreased during 4 h the copy number of the lowest number of copies of 10 μg/m L, 20 μg/m L, 40 μg/m L r SPLUNC1 concentrations than the control group reduced 2.96%(p>0.05), 92.57%(p<0.01), 93.75%(p<0.01). Studies have shown that, r SPLUNC1 of Mo in vitro significantly inhibited.(3) r SPLUNC1 protein significantly suppressed Pasteurella, growth of E. coli, a dose effect, and Streptococcus pneumoniae, Staphylococcus aureus no growth inhibitory effect. Description r SPLUNC1 can inhibit certain pathogenic bacteria, especially gram-negative bacteria have a strong inhibitory effect.(4) 10μg/ml r SPLUNC1 significantly improve Mo infection of the outer peripheral neutrophils NE activity(p <0.05), 20 ~ 40μg / ml r SPLUNC1 significantly improved Mo infection of the outer peripheral neutrophils NE activity(p<0.01, p<0.01). Description r SPLUNC1 can be adjusted NE activity against respiratory infections is important.(5)Fluorescent staining apoptosis have no green fluorescence signal, agarose gel electrophoresis is also no apoptosis characteristic DNA Ladder bands.Description SPLUNC1 pathogen infection did not induce apoptosis of lymphocytes.