The Replication Of Infectious Spleen And Kidney Necrosis Virus And Siniperca Chuatsi Rhabdovirus Inhibited By Tumor Suppressor Gene P53 In Siniperca Chuatsi

The Chinese perch or mandarin fish Siniperca chuatsi is one of the major cultured fish with a high value in China.With the increase of breeding density,disease problems become more and more serious.Among them,infectious kidney and spleen necrosis virus disease(ISKNVD)and S.chuatsi rhabdovirus disease(SCRVD)have posed a serious threat to the healthy development of mandarin fish farming industry.Therefore,it is very necessary to carry out research on prevention of ISKNV and SCRV.P53 is an important tumor suppressor gene,which regulates cell cycle,controls cell death and differentiation,participates in apoptosis and other functions.In recent years,researcher discovered that p53 protein played an important role in the innate antiviral response.It has become a new hot spot for cell innate immunity and virus immune escape in the field of virus control.In this study,the p53 gene in mandarin fish was cloned and expressed and the polyclonal antibody was prepared.And the response pattern of ISKNV and SCRV infection and its antiviral function were studied.The results obtained in this study were as follows:1.The p53 sequence in mandarin fish was obtained and its polyclonal antibody was prepared.The response pattern of p53 to ISKNV and SCRV infection was discussed.The full-length cDNA of p53(Sc-p53)is 1,555 bp and encodes 380 amino acids.The 5' and 3' non-coding regions are 165 bp and 247 bp,respectively.The sequence showed the closest homology with the p53 protein of Stegastes partitus.Sequence comparison of the Sc-p53 protein with p53 proteins from evolutionarily distant species showed that the Sc-p53 protein shares 5 amino acid clusters with other p53 proteins.The prokaryotic expression vector p ET30a-Scp53 was constructed and transformed into Escherichia coli BL21.The induction of IPTG induced the expression of Sc-p53,and the Sc-p53 protein existed as inclusion bodies.The purified Sc-p53 recombinant protein was immunized in rabbits to prepare rabbit anti-Sc-p53 polyclonal antibody.Quantitative real-time PCR assays revealed that Sc-p53 was expressed in all tissues examined,and it was most abundant in the gill and kidney.And western blotting revealed that Sc-p53 was expressed in liver,gill,spleen,stomach and heart.In addition,the regulation of Sc-p53 gene expression after experimental viral infection was determined and characterized.The m RNA and protein expression of Sc-p53 were significantly up-regulated in the Chinese perch brain(CPB)cell line and mandarin fish after infection with ISKNV.The expression of Sc-p53 in the spleens of the ISKNV infected mandarin fish was higher than that in the control fish at 3 h post-infection.The level of Sc-p53 expression then dropped,but increased significantly again from 48 h to 7 d post-infection.The profile of Sc-p53 expression in CPB cells showed a similar pattern as in spleens.The results of western blotting showed that the p53 level of ISKNV infected with CPB also showed a biphasic increase.However,a different expression pattern of Sc-p53 in response to SCRV infection was found.The m RNA expression of Sc-p53 was significantly up-regulated in CPB at 6 h and spleen of mandarin fish at 24 h post-infection.The protein expression of Sc-p53 was significantly up-regulated in CPB at 1 h,remained elevated at 4 h,and then decreased to control level by SCRV.2.The organization of mandarin fish p53 genome sequence was determined and a novel splice variant(Sc-p53I6)was characterized.And then the response mode of Sc-p53I6 to ISKNV and SCRV infection was further discussed.The Sc-p53 genomic sequence was composed of 5,543 bp,containing 11 exons and 10 introns,which was similar to other species.The full-length cDNA of a novel splice variant p53 from mandarin fish(designed as Sc-p53I6)is 1,106 bp and encodes 254 amino acids.The 5' and 3' non-coding regions are 165 bp and 179 bp,respectively.Compared to Sc-p53,the full-length cDNA of Sc-p53I6 lacks five exons and retains part of the sixth intron.Quantitative real-time PCR assays revealed that Scp53I6 was expressed in all tissues examined,and it was most abundant in the gill,hemocyte and hind kidney.Western blotting analysis revealed that Sc-p53I6 was abundant in liver,trunk kidney,hind kidney,stomach and heart.In addition,the regulation of Sc-p53I6 transcriptional expression after virus infection was determined and characterized.The results showed a biphasic increase expression pattern of Scp53I6 in spleen of mandarin fish following ISKNV challenge.The expression of Sc-p53I6 in the spleens of the ISKNV infected mandarin fish was higher than that in the control fish at 6 h post-infection.The level of Sc-p53I6 expression then dropped,but increased significantly again from 48 h to 72 h post-infection.However,a different expression pattern,once rise,of Sc-p53I6 in response to SCRV infection was found.The m RNA expression of Sc-p53I6 was significantly upregulated in CPB at 4 h and spleen of mandarin fish at 12 h post-infection.3.In order to study the antiviral function of Sc-p53,Sc-p53 knockdown CPB cell line was constructed.And the effects of Sc-p53 knockdown CPB cell line on the replication of ISKNV and SCRV were further explored.First of all,the RANi eukaryotic expression vectors,p MAGic-Scp53 A and p MAGic-Scp53 B were constructed.The vectors were transfected onto CPB to obtain stable transfected cell lines,designated as CPB-p53kd-A and CPB-p53kd-B.To examine effect of Sc-p53 knockdown efficiency,quantitative real-time PCR and western blotting were performed on CPB-p53kd-A and CPB-p53kd-B with wild type CPB cells.The results showed that the m RNA levels of Sc-p53 were reduced by 63% and 60%,the protein levels of Sc-p53 were reduced by 79.42% 78.28% in the CPB-p53kd-A and CPB-p53kd-B group,respectively.These results indicated that two p53 knockdown CPB stable cell lines in mandarin fish were successfully constructed.Then ISKNV and SCRV were used to infect the Sc-p53 knockdown CPB cell lines.The results showed that the copy number of ISKNV and SCRV and the expression of ISKNV-MCP and SCRV-N protein were significantly increased in CPB-p53kd-A and CPB-p53kd-B compared with the control group and the increased level was positively correlated with knockdown efficiency.4.The replication of ISKNV and SCRV inhibited by promoting the expression of Sc-p53 protein.The expression of Sc-p53 protein enhanced by addition of 5-fluorouracil.Compared with the control group,the virus copy number of ISKNV and SCRV and the expression of ISKNV-MCP and SCRV-N protein in the 5-fluorouracil group were significantly decrease after ISKNV and SCRV challenge.In summary,the replication of ISKNV and SCRV in the Sc-p53 knockdown CPB cell line was significantly increased.And after the promotion of Sc-p53 expression,the replication of ISKNV and SCRV was significantly decreased.It was suggested that Sc-p53 play an important antiviral effect in the replication of ISKNV and SCRV.