Cloning Procine TRIM11 And Studying Its Effect Of PRV Replication

The TRIM proteins are members of a large family of proteins characterized by their shared tripartite motif structure,also known as the RBCC domain.TRIM proteins are involved in diverse cellular processes,including cell proliferation,differentiation,oncogenesis,and apoptosis.A lot of TRIM proteins evolved as components of innate immunity or exert their immune function by directly interacting with virus components to regulate infection course of virus.TRIM11 is a member of the TRIM family proteins.Human TRIM11 could suppress infectivity of HIV-1,by suppressing virus entry,gene transcription and release.However,Human TRIM11 inhibits IRF3 activation,reducing the IFN-induced antiviral state against HSV-1.Therefore,TRIM11 represent the function of multiform regulation in the course of virus infection.Presently,study about porcine TRIM11 is very few,this research established the PK15 cell line expressing porcine TRIM11 stably,provided the experimental basis for research on the porcine TRIM11 biological activity in the future.In order to study porcine TRIM11 influence on PRV infection.The TRIM11 c DNA was cloned from the total c DNA of porcine submaxillary lymph nodes.Then analysed TRIM11 c DNA sequence by using bioinformatics,incluing tissue expression profile analysis and phylogenetic analysis.In order to obtain the stable transfection cells,TRIM11 c DNA was connected into Piggy Bac(PB)transposon vector,then the recombinant vector(PB-TRIM11)was transfected to PK15 cells.The stable transfection cells were screened by puromycin,and the expression of TRIM11 was determined by q RT-PCR.Infected PK15 cell line that stably express porcine TRIM11 and control cells with 0.1 MOI PRV,respectivly.Harvested cell supernatant and detected the viral TCID50 at 12 h,24 h,36 h after infection.Infected PK15 cells with 0.1 MOI PRV.Harvested cells and total RNA was extracted to obtain c DNA by reverse transcription-PCR at 12 h,24 h,36 h after infection.Detected the change of TRIM11 expression at transcriptional level.Infected PK15 cell line that stably express porcine TRIM11 and control cells with 0.1 MOI PRV,respectivly.Meanwhile,the other groups which PK15 cell line that stably express porcine TRIM11 and control cells didn?t infect with PRV served as blank control.Harvested cells and total RNA was extracted to obtain c DNA by reverse transcription-PCR at 12 h after infection.Detected the change of the IFN-? expression at transcriptional level.Transfected PK15 cell line that stably express porcine TRIM11 and control cells with IFN-?-Luc vector.Infected the two cell groups with 0.1 MOI PRV.Meanwhile,the other two cell groups didn?t infect with PRV served as blank control.Harvested cells at 12 h after infection.Detected the change of TRIM11 promoter activity by Dual-luciferase reporter gene assay.The results showed that the coding length of the cloned porcine TRIM11 is 1 407 bp and the sequence code 468 amino acids.The molecular weight of the protein coded by TRIM11 is 53 k Da,its isoelectric point is 5.409.TRIM11 protein is characterized by the presence of the tripartite motif consisting of a RING domain,two B-box motifs followed by a coiled-coil region,it possess a PRV domain and a SPRY domain at its C-terminus.Tissue expression profile analysis showed that,the expression of porcine TRIM11 was higher in fat?lung,whereas the expression of porcine TRIM11 was lower in heart?ileum?recutum?duodenum.DNA sequence analysis of TRIM11 showed that the cloned TRIM11 c DNA was consistent with sequence from Gen Bank and the recombinant vector(PB-TRIM11)was constructed.After screening culture by puromycin,stable transfected PK15 cell line was established and the expression of TRIM11 was identified by q RT-PCR.Harvested cell supernatant and detected the viral TCID50 after infected stably express porcine TRIM11 and control cells with PRV,the results showed that the viral TCID50 of the TRIM11 over-expression group was higher than the control all long.The diffence between titer of virus on infected stably express porcine TRIM11 and control cells was significant(P<0.05)after infected 12 h.It indicated that the TRIM11 overexpression cells might be more susceptible to PRV.TRIM11 expression at transcriptional level was inhibited in PK15 cells after infected with PRV.TRIM11 overexpression promoted IFN-? expression during PRV infection.