Study On The Role Of Host Protein SERBP1 In The Apoptosis Of Goat Primary Turbinate Bone Cells Induced By Orf Virus

Contagious pustular dermertitis,also known as"Orf virus",is an acute,contagious,epitheliotropic infectious disease caused by ovine aphthous virus（ORFV）,which not only causes serious economic losses,but also threatens human health.The protein CGI-55,also known as SERBP1 protein,its overexpression inhibits apoptosis caused by ER stress.Yeast two-hybrid results showed that SERBP1 interacted with ORFV125,and ORFV125 inhibited apoptosis.Previous studies by our group have found that ORFV can induce the apoptosis of goat primary turbinate bone cells,the mechanism of SERBP1 protein in ORFV-induced apoptosis is still unclear.In this study,the expression of SERBP1 gene was overexpressed or knocked down on the basis of cloning the CDS region of SERBP1 gene in goats.The effect of SERBP1protein on apoptosis and expression of related genes was preliminarily verified.Meanwhile,on the basis of overexpression and knockdown of SERBP1 gene,the role of SERBP1 protein in ORFV-induced apoptosis of goat turbinate primary cells was studied,laying a foundation for further study on the mechanism of SERBP1 protein in ORFV-mediated inhibition of host cell apoptosis.The results are as follows:1.Cloning of CDS region of SERBP1 gene and construction of eukaryotic expression vector in goatSpecific primers were designed according to the predicted sequence of goat SERBP1 gene in GenBank（XM＿018045378.1）,and the CDS region of SERBP1 gene was amplified using goat spleen c DNA as template.The sequence was identified and analyzed.Meanwhile,the CDS region of SERBP1 gene was cloned into pcDNA3.1（+）vector to construct eukaryotic expression vector.Results showed that SERBP1 gene was successfully amplified with 1293 bp,containing 5’UTR 44 bp,CDS region 1179bp,3’UTR 70 bp,encoding 392 amino acid residues,and the eukaryotic expression vector pcDNA3.1（+）-SERBP1 was successfully constructed.Bioinformatics analysis showed that the protein was a stable hydrophobic protein with no signal peptide and no transmembrane domain.SERBP1 is mainly located in the nucleus and contains two phosphorylation sites.It is predicted that SERBP1 may interact with 10 host proteins,including RPL31 and RPS3.2.Effects of overexpression of goat SERBP1 gene on the proliferation,cell cycle,apoptosis and apoptosis-related genes of goat primary turbinate bone cells（1）Primary culture of goat primary turbinate bone cells and expression of SERBP1 protein in cellsNasal turbinates were collected from newly born non-lactating healthy goats,and goat primary turbinate bone cells were isolated and cultured using 1.3 mg/m L collagenase I,1.3 mg/m L collagenase IV,0.125%trypsin digestion and tissue block methods,respectively.The results showed that compared with enzymatic digestion method,the number of cells obtained by tissue block method was higher,the purity was higher,the cell proliferation ability was better,and it could be continuously transmitted to more than 70 generations.pcDNA3.1（+）-SERBP1 was transfected into goat primary turbinate bone cells,and Western blot demonstrated that SERBP1protein was correctly expressed in goat primary turbinate bone cells,as expected.（2）Effects of overexpression of SERBP1 gene on proliferation,cycle,apoptosis and apoptosis-related genes of goat primary turbinate bone cellsAfter transfection of pcDNA3.1（+）-SERBP1 and pcDNA3.1（+）vectors into goat primary turbinate bone cells for 48 h,the effects of overexpression of SERBP1 on cell proliferation were detected by MTT assay,and the effects of overexpression on cell cycle and apoptosis were detected by flow cytometry.The results showed that overexpression of SERBP1 gene could promote the proliferation and apoptosis of cells,and the cells were arrested in G2/M phase;it could also up-regulate the expression of Caspase3,Caspase7,P53 and PARP1 gene mRNA levels.3.Effects of SERBP1 gene knockdown on proliferation,cycle,apoptosis and apoptosis-related genes of goat primary turbinate bone cells（1）Interference with the synthesis of siRNA fragmentsThree siRNA interference fragments were designed and synthesized according to the sequence of SERBP1 gene.After the three siRNA interference fragments were transfected into goat primary turbinate bone cells for 48h,RT-q PCR detection results showed that SERBP1-siRNA1 had the best interference effect on SERBP1.The mRNA expression level of SERBP1 gene was reduced by 70%.（2）Effects of knockdown SERBP1 gene on proliferation,cycle,apoptosis and apoptosis-related genes of goat primary turbinate bone cellsAfter transfection of SERBP1-siRNA1 and Negative Control into goat primary turbinate bone cells for 48 h,the effect of knockdown of SERBP1 on cell proliferation was detected by MTT assay,and the effect of knockdown of SERBP1 on cell cycle and apoptosis was detected by flow cytometry.The results showed that knockdown of SERBP1 gene inhibited cell proliferation and apoptosis,and the cells were arrested in G0/G1 phase;it could also down-regulate the expression of Caspase3,Caspase7,BCL2L11 and Bax gene mRNA.4.Study on the role of SERBP1 protein in ORFV-induced apoptosis of goat primary turbinate bone cellsFlow cytometry was used to detect the effect of 100 TCID₅₀ ORFV on apoptosis after infection of goat primary turbinate bone cells,and the results showed that ORFV could induce apoptosis of goat turbinate osteocytes.RT-q PCR was used to detect the expression of SERBP1 gene in goat primary turbinate bone cells at different time points（12 h,24 h,36 h,48 h,60 h and 72 h）of ORFV infection,and the results showed that ORFV could promote SERBP1 gene expression,with the highest expression at 48 h.To further analyze the role of SERBP1 during ORFV infection,ORFV was used to infect goat primary turbinate bone cells overexpressing and knocking down SERBP1,respectively,to detect its effect on ORFV-induced apoptosis and apoptosis-related gene expression,and knocking down SERBP1 gene inhibited ORFV-infected apoptosis,resulting in decreased expression of intracellular pro-apoptotic genes Caspase3 and Caspase7 mRNA and increased expression of anti-apoptotic gene Bcl-2 mRNA;overexpressing SERBP1 gene promoted apoptosis of ORFV-infected cells,resulting in increased expression of intracellular Caspase7mRNA and decreased expression of Bcl-2 mRNA.