Partial Biological Functions Of Sheep SP-A Gene In Vitro

AIM:Based on the expression and purification of SP-A gene in bashiby sheep and Argal hybird sheep,the effects of recombinant SP-A protein?rSP-A?on the proliferation and metabolism of Mycoplasma ovipneumoniae?MO?cultured in vitro,on the adhesion of MO,and on the killing effect of eosinophils?EOS?on MO were studied.To lay a foundation for studying the biological function of rSP-A in vitro.Methods:?1?Expression and protein purification of SP-A gene:Recombinant Pasteurella pastoris GS115/pPIC9K-SP-A positive clone was constructed from bashiby sheep and Argal hybird sheep.Methanol was used to induce expression for 96 hours.Ni-NTA agarose affinity chromatography was used to purify the SP-A gene.SDS-PAGE electrophoresis was used to detect the target protein.?2?The effect of rSP-A on the proliferation of MO:The rSP-A of bashiby sheep and Argal hybird sheep at different concentrations?10?g/mL,20?g/mL,40?g/mL?was added to MO culture medium for in vitro culture.The number of MO colonies was observed by plate colony counting method,and the expression of MO 16S rRNA was detected by real-time fluorescence quantitative PCR.?3?Effects of rSP-A on MO metabolism:rSP-A of sheep with different concentrations was added to MO culture medium for in vitro culture.The culture medium was collected on 1,3,5 and 7 days,and the absorbance(OD₅₅₀)at 550 nm was measured by acidification method.?4?The effect of rSP-A on the adhesion of MO:rSP-A of sheep with different concentrations was added to the process of adhesion between MO and TC-1 cells.Adhesion rate was detected by 50%color change unit method,and the expression of adhesin P113 gene was detected by real-time fluorescence quantitative PCR.?5?The effect of rSP-A on EOS killing MO:Eosinophils in sheep peripheral blood were isolated by discontinuous density gradient centrifugation.After infection with MO,rSP-A with different concentrations was added to the plate for 30 minutes,and then cultured in vitro for 7 days.Colony counting method was used to count the number of colony after infection.Results:?1?SDS-PAGE assay showed the target band at 26.38 kDa after methanol-induced eukaryotic vector expression.After purification,the impurity protein was greatly reduced,and only a single band with a molecular weight of 26.38 kDa appeared,which was consistent with the expected molecular weight of the protein.This indicated that the rSP-A expression of Bashiby sheep and Argal hybird sheep was successful and the purification effect was good.?2?The results of plate colony counting showed that the number of colonies in the bashiby sheep 10?g/mL,20?g/mL and 40?g/mL groups decreased by 8.35%?P>0.05?,23.04%?P<0.05?and 40.03%?P<0.01?,respectively,compared with the control group,while the number of colonies in the Argal hybird rSP-A group decreased by 6.73%?P>0.05?,21.74%?P<0.05?and 37.11%?P<0.01?,respectively.The results of quantitative PCR showed that the number of copies of MO 16S rRNA decreased to the lowest level 4 hours after adding rSP-A.The number of copies of the three rSP-A concentration groups of bashiby sheep was 28.85%?P<0.05?,22.19%?P<0.01?,18.51%?P<0.01?,and that of the three rSP-A concentration groups of disc sheep was 68.16%?P>0.05?,28.65%?P<0.01?,20.95%?P<0.01?of the control group.?3?The absorbance value at 550 nm was measured.The results showed that at 1 day,the absorbance value of 20?g/mL group was significantly lower than that of the control group?P<0.05?,and that of 40?g/mL group was significantly lower than that of the control group?P<0.01?.From the 3rd day,the OD value of each experimental group was significantly lower than that of the control group?P<0.01?.On the7th day,the OD value of 40?g/mL of Bashiby sheep and Argal hybird sheep was 37.6%lower than that of the control group 35.7%?P<0.01?.?4?50%color change unit method showed that the adhesion rate of MO in 40?g/mL group of Bashiby sheep and Argal hybird sheep was 5.93%?P<0.05?and 6.60%?P<0.05?lower than that of the control group,respectively.Real-time q-PCR showed that the expression of mRNA of adhesion P113 gene in 40?g/mL group of bashiby sheep and Argal hybird sheep was 23.7%?P<0.01?and 22.9%?P<0.05?lower than that of the control group,respectively.?5?The results showed that the killing rate of 10?g/mL group was decreased by 5.6%?P<0.05?and 6.1%?P<0.05?,12.4%?P<0.01?and 12.6%?P<0.01?respectively,and 19.8%?P<0.01?and 20.4%?P<0.01?respectively.Conclusion:?1?rSP-A of two kinds of sheep significantly inhibited the proliferation of MO in vitro.?2?rSP-A could significantly reduce the metabolic rate of MO in vitro.?3?rSP-A could significantly inhibit the adhesion of MO to Bashiby sheep and Argal hyvird sheep at a higher concentration?40?g/mL?.?4?rSP-A could significantly inhibit the killing effect of EOS on MO.