Construction Of JSRV-Env Lentiviral Vector And The Effect Of JSRV Env On The Expression Of SUN2

Ovine pulmonary adenocarcinoma(OPA)is a neoplastic disease caused by Jaagsiekte sheep retrovirus(JSRV).At present,there is no in vitro culture system for JSRV,and conventional virus isolation methods cannot obtain live viruses,which greatly restricts the study of the disease.Through the analysis of RNA-Seq(PRJEB27638)data of artificially infected JSRV,our team found that the expression of SAD1/UN84 domain protein-2(SUN2)decreased during JSRV infection,suggesting that SUN2 may interact with virus and participate in important biological processes such as cell proliferation and differentiation during JSRV infection.Therefore,this study carried out the following experiments:In this study,a lentiviral overexpression vector of envelope protein(Env)of sheep pulmonary adenoma virus(JSRV-NM)was constructed to infect human bronchial epithelial cells-2b(BEAS-2B).The BEAS-2B cell line with stable expression of Env was screened and the effects of Env on cell proliferation,migration and invasion were detected.The lung tissue of sheep with OPA natural cases was used as the experimental group,and the lung tissue of healthy sheep was used as the control group.Quantitative Real-time PCR(q PCR)and Western Blot were used to detect the relative expression of SUN2 at the transcriptional level and protein level in 6 tissue samples.BEAS-2B cells infected with JSRV-env lentivirus were used as the experimental group,BEAS-2B cells infected with negative lentivirus were used as the negative control group,and BEAS-2B cells without treatment were used as the blank control group.The relative expression of SUN2 at the transcriptional and protein levels was detected by q PCR and Western Blot.The results showed that the env gene was successfully amplified and inserted into the PMT194 vector.The enzyme digestion identification was consistent with the expectation,indicating that the PMT194-JSRV-env lentiviral expression vector was successfully constructed.After transfection of 293T cells for 72 h,the expression of env was significantly higher than that of blank control group and negative control group(P<0.001).The average titer of JSRV-env recombinant lentivirus was 1.03×10~9 IU/m L.The optimal multiplicity of infection(MOI)of BEAS-2B cells infected with lentivirus was 40,and the optimal Puro concentration for screening cell lines was 0.3μg/m L.Fluorescence microscopy showed that BEAS-2B cells expressed red fluorescence.The results of q PCR and Western Blot showed that the expression of Env in the experimental group was significantly higher than that in the blank control group and the negative control group(P<0.001).The Env-FLAG fusion protein was successfully expressed in the host cells.Compared with the blank control group and the negative control group,the proliferation and migration ability of the cells in the experimental group were significantly enhanced(P<0.05).Compared with the healthy sheep lung tissue,the expression of SUN2 m RNA and protein in the lung tissue of OPA natural cases was significantly lower than that in the control group(P<0.01).Compared with the negative control group and the blank control group,the expression of SUN2 m RNA and protein in the experimental group was significantly lower than that in the control group(P<0.01).In summary,this study successfully constructed JSRV-env overexpression lentiviral vector;JSRV Env overexpression BEAS-2B cell line was successfully screened.The expression of SUN2 was significantly decreased in the lung tissue of OPA natural cases and JSRV Env overexpression BEAS-2B cell line.