Study Of Recombinant SPLUNC1 Against Mycoplasma Ovis Pneumoniae In Vitro

AIM:Mycoplasma ovipneumoniae（MO）is a mixed infectious disease of sheep,and its main pathogen is Mycoplasma ovipneumoniae（MO）.The spread of the disease can cause economic crisis in sheep farms.In this study,sheep recombinant SPLUNC1 protein（rSPLUNC1）was used to investigate its anti-MO function in vitro.Firstly,the differences of SPLUNC1 gene expression levels in different tissues and organs of different breeds of sheep were compared.The effects of rSPLUNC1 on the metabolism of MO cultured in vitro,the adhesion of MO to goat tracheal epithelial cells,the killing effect of EOS on MO,and the effect ofα1-antitrypsin on the regulation of rSPLUNC1 against MO were studied.The role of this protein in the anti-MO infection was analyzed and verified,providing new materials for the prevention and treatment of Mycoplasma ovis pneumonia.Methods:（1）Expression of SPLUNC1 gene and purification of SPLUNC1 protein:The positive Pichia pastoris strain GS115/p PIC9K-SPLUNC1 preserved by our team was induced and purified,and the target protein was verified by SDS-PAGE electrophoresis.（2）Sequence comparison and quantitative analysis of SPLUNC1 gene in different sheep breeds:The full-length c DNA of SPLUNC1 gene in Altay sheep and Suffolk sheep were cloned,and the sequence differences were compared.RNA was extracted from 9 tissues and organs of the two sheep by Trizol method,and c DNA was synthesized by reverse transcription.The m RNA copy number of SPLUNC1 in each tissue and organ of the two sheep was detected by Real-time PCR.（3）Effect of rSPLUNC1 on MO metabolism:rSPLUNC1 with gradient concentration（10μg/m L,20μg/m L,40μg/m L）was added to the MO culture medium for co-culture.The test solution was collected on the 1st,3rd,5th and 7th days,respectively.The absorbance of the test solution was detected by acidification method at 550 nm.（4）Effect of rSPLUNC1 on MO adhesion cells:Rabbit anti-MO antibody was used as the primary antibody,and goat anti-rabbit Ig G labeled with FITC was used as the secondary antibody.The fluorescence intensity of rSPLUNC1 with gradient concentrations（10μg/m L,20μg/m L,40μg/m L）on MO adhesion goat tracheal epithelial cells was detected by indirect immunofluorescence method.（5）The effect of rSPLUNC1 on EOS killing MO:Eosinophils from sheep peripheral blood were isolated by discontinuous density gradient centrifugation.After 10⁶CCU/m L MO infection,rSPLUNC1 with gradient concentrations（10μg/m L,20μg/m L,40μg/m L）was added to co-culture,and then transferred into PPLO plate for 7 days.The colony counting method was used to calculate the killing rate of each group.（6）The effect ofα1-antitrypsin on anti-MO of SPLUNC1 protein:10⁶CCU/m L MO was added to the isolated sheep neutrophils to stimulate the release of neutrophil protease（NE）.The substrate color method was used to detect the change of NE activity under the specific concentration of rSPLUNC1（10μg/m L）and different concentrations ofα1-antitrypsin（10μg/m L,20μg/m L,40μg/m L）.At the same time,the copy number of MO 16 s rRNA was detected by Real-time PCR.Results:（1）After induced by methanol for 72h,the target band was detected by SDS-PAGE electrophoresis at 25.53k Da,and there was no impurity protein after purification.（2）The full-length c DNA of SPLUNC1 gene in Altay sheep and multifetal Suffolk sheep was 768 bp,and the nucleotide sequence similarity was 99.87%.The expression of SPLUNC1 m RNA in 9 tissues and organs of Altay sheep was significantly higher than that of Suffolk sheep（p<0.05）.（3）The absorbance at OD₅₅₀nm of the culture medium was detected.The results showed that with the increase of protein concentration,the OD₅₅₀value decreased gradually,and the MO metabolic rate decreased continuously.At 1d,compared with the control group,the metabolic rate of MO in the medium and high concentration groups decreased by 13.23%and 19.13%respectively（p<0.05）.At 3d,the metabolic rate of MO in the low,medium and high concentration groups was significantly different from that in the control group（p<0.05）.At 7d,the metabolic rate of MO in the concentration range of 10-40μg/m L rSPLUNC1 decreased by 11.20%,24.51%and 35.86%respectively（p<0.05）.（4）Indirect immunofluorescence showed that after 10μg/m L rSPLUNC1 acted on goat tracheal epithelial cells,a large amount of green fluorescence was produced.With the increase of protein concentration,the fluorescence gradually decreased and the fluorescence intensity gradually weakened.After 40μg/m L rSPLUNC1 acted on cells,the fluorescence was the smallest and the intensity was the weakest.（5）Compared with the control group,the killing rate of medium and high rSPLUNC1 concentration groups decreased by 9.2%and17.7%（p<0.05）.（6）The NE activity was detected.The results showed that with the increase ofα1-AT concentration,the NE activity decreased continuously,showing a concentration-dependent relationship withα1-AT.Compared with the control group,the NE activity at medium and high concentrations ofα1-AT decreased by 38.58%and 71.18%,respectively（p<0.05）.The expression of MO 16s rRNA was detected.Compared with the control group,10-40 ug/m Lα1-AT could reduce the copy number of MO 16s rRNA to varying degrees,and the copy numbers were reduced by 34.2%,82.6%and 97.3%,respectively（p<0.05）.Conclusion:（1）The expression of sheep rSPLUNC1 was successful and the purification effect was good.（2）The distribution of sheep SPLUNC1 gene was tissue-specific,and the expression of SPLUNC1 m RNA in the trachea of the two breeds was the highest.（3）rSPLUNC1 could inhibit the metabolism of MO in a concentration-dependent manner.（4）Different concentrations of rSPLUNC1 inhibited the adhesion of MO to goat tracheal epithelial cells,and the inhibitory effect of high concentration of rSPLUNC1 was the most obvious.（5）rSPLUNC1 can inhibit the killing of EOS on MO,showing a concentration-dependent effect.（6）The regulation ofα1-AT on MO is mediated by SPLUNC1.