Study On Function In Ubiquitin-like ISG15Gene Of Argali,bashibai Sheep And Their Hybrid Sheep

Argali (Ovis ammon) is the second class protected animal in china, has a strong disease resistance, however, its hybrid sheep showed weakly disease resistance, Speculating that the weak disease resistance of the hybrid sheep may be associate with the variation of some resistance genes. Ubiquitin-like gene ISG15is a kind of disease resistance gene, plays an important role in the body’s innate immunity. Therefore, speculate the decrease of the innate immune of the hybird sheep presumably due to the variation of ISG15gene.Object:This study through the expression of ISG15gene of Argali,Bashibai sheep and their hybrids sheep to study the recombinant ISG15protein effect on lymphocyte proliferation, apoptosis and mycoplasma infection in vitro, compared the different expression level of ISG15between the parents and hybrid sheep, in order to reveal the function of the ubiquitin-like protein ISG15, to clarify its role in disease resistance.Methods:(1) The eukaryotic expression of ISG15gene of Argali, Bashibai sheep and their hybrid sheep:First the ISG15gene was amplified from recombinant cloning vector pMD18-T-ISG15, then the ISG15gene of the three sheep were subcloned into the Pichia pastoris pPIC9K expression vector, the recombinant plasmid was transformed by electroporation into Rubaside Pichia pastoris GS115strain, and the positive transformants were identified by using PCR. Then used the0.5%methanol to induce the expression of the protein in the recombinant strain GS115/pPIC9K-ISG15; finally verify the protein expression by SDS-PAGE.(2) The purification of ubiquitin-like ISG15gene expression product of Argali, Bashibai sheep and their hybrid sheep:The reorganization ubiquitin-like ISG15protein of the three sheep were purified by Ni2+chelate affinity chromatography, then the purified product was detected by SDS-PAGE.(3) The recombinant ubiquitin-like ISG15protein of Argali, Bashibai sheep and their hybrid sheep effect on lymphocyte proliferation:The jugular venous blood was collected from the healthy wool sheep, lymphocytes were separated by using lymphocyte separation medium, cultured the cells, the purified recombinant ubiquitin-like ISG15protein of the three sheep were added to the cultured cells, the effect of the recombinant ubiquitin-like ISG15protein on the lymphocytes proliferation was detected by MTT.(4) The effect of recombinant ubiquitin-like ISG15protein of Argali, the Bashibai sheep and their hybrid sheep on lymphocyte apoptosis:Cultured sheep lymphocytes, then the genomic DNA was extracted by using reagents, the apoptosis was detected by agarose gel electrophoresis method.(5) The effect of recombinant ubiquitin-like ISG15protein of Argali, Bashibai sheep and their hybrid sheep on mycoplasma infection in vitro:Cultured sheep lymphocytes, and then respectively added the recombinant ubiquitin-like protein ISG15of the three sheep and Mycoplasma ovipneumoniae into the cultured lymphocytes, used real-time PCR technology to detect the expression level of Mycoplasma ovipneumoniae16S rRNA.(6) Detected the expression level of ubiquitin-like ISG15mRNA of Argali, Bashibai sheep and their hybrid sheep in vitro: Cultured sheep lymphocyte, then infected with Mycoplasma ovipneumoniae, used real-time PCR technology to detect the expression level of ISG15mRNA.Results and Conclusion:(1) We successfully constructed ISG15eukaryotic expression vectors of the Argali, Bashibai sheep and their hybrid sheep, and successfully expressed in Pichia pastoris.(2) The purified products were analysed by SDS-PAGE, strip single target band could be observed, the recombinant ubiquitin-like ISG15protein of the Argali, Bashibai sheep and their hybrid sheep were successfully purified.(3) when added the recombinant ISG15protein of Argali concentrations of5,10,20,40and80μg/mL, the OD value was significant higher than control group(P<0.05); when added the recombinant ISG15protein of the Bashibai sheep concentrations of40and80μg/mL, the OD value was significant higher than control group(P<0.05); however, when added the recombinant ISG15protein of hybrid sheep concentrations of10,20,40and80μg/mL, the OD value was significant lower than control group(P<0.05). The results showed recombinant ISG15protein of Argali and Bashibai sheep could well promote lymphocyte proliferation activity, while the recombinant ISG15protein of hybrid sheep had poorly promoted lymphocyte proliferation activity.(4) Agarose gel electrophoresis showed that the ladder did not appear, The results showed recombinant protein of Argali, the Bashibai sheep and their hybrid sheep had no effect on promoting the lymphocyte apoptosis.(5) When added the recombinant ubiquitin-like protein ISG15from12to96hour, the expression levels of Mycoplasma ovipneumoniae16S rRNA in Argali and Bashibai sheep group were significant lower than control goup(P<0.05), while the hybrid sheep group had no significant with control goup. The results showed recombinant ubiquitin-like protein ISG15of Argali and Bashibai sheep could inhibit the grouth of Mycoplasma ovipneumoniae at12hour, while the reorganization of ubiquitin-like protein ISG15of hybrid sheep no significant inhibitory effect on Mycoplasma ovipneumoniae.(6) Mycoplasma ovipneumoniae challenged from12to96hours, the ubiquitin-like ISG15mRNA expression level of Argali and Bashibai sheep experimental groups were significant higher than their control goups and hybrid sheep experimental group(P<0.05). The ISG15mRNA expression levels of lymphocyte in Argali and Bashibai sheep could significantly improve by Mycoplasma ovipneumoniae challenged, while The ISG15mRNA expression levels of lymphocyte in hybrid sheep had no significantly improved.