Transcriptome Analysis Of Liver And Spleen Of Takifugu Rubrripes And Recombinant Preparation Of IL-2 And IFN-?

As an important marine economic fish,Takifugu rubrripes are becoming more and more industrialized with the rapid development of its aquaculture industry,which make Takifugu rubrripes vulnerable to a variety of pathogens.Therefore,it is of theoretical significance and practical value to study the transcriptome of immune tissue,develop natural immune genes and their recombinant expression.The content of this research was divided into two parts.In the first part,RNA-Seq was used to analyze the transcriptome of liver and spleen tissues of male and female Takifugu rubrripes,and the expression of some functional genes and the differential expression among different samples were analyzed.The results showed that the Clean Data of the four transcriptional groups were 24,236,217?28,316,186?44,052,729 and 32,624,842 respectively,including liver tissue of male fish(mL),liver tissue of female fish(fL),spleen tissue of male fish(mS)and spleen tissue of female fish(fS).In the differentially expressed gene analysis,551 differentially expressed genes were screened in the mL VS fL differential group;293 differentially expressed genes were detected in the mS VS fS differential group;3,830 differentially expressed genes were obtained from the mL VS mS difference group.During the fL VS fS comparison,a total of 3969 differentially expressed genes were detected.GO enrichment analysis of differentially expressed genes was carried out in four different groupings.According to GO analysis and NR data annotation,it was found that some immune-related genes play important regulatory roles in immune tissues represented by spleen and liver,such as interferon and its regulatory factors(Ifi44l,Irf3,Irf4,Ifn-?),interleukin and its receptor genes(l1rl1,Il2,Il6 r,Il7ra,Il8,Il12b?Il21r)and vitellogenin gene(Vtg-1)etc.In addition,KEGG enrichment analysis of differentially expressed genes showed that the differentially expressed genes were mainly concentrated in cytokine-cytokine receptor interaction,Toll-like signaling pathway and RIG-I receptor signaling pathway during the comparison between male and female Takifugu rubrripes.They are mainly involved in the process of cell growth and differentiation,and can also mediate the production of immune cells and induce their innate and adaptive immune responses.In the second part,IFN-? protein products were produced by genetic engineering bacteria G-IFN-?,which had been constructed in the laboratory.The genetic engineering bacteria G-IFN-? were fermented in 5 L fermentation tank.YPD and BMGY medium were used as fermentation broth.The parameters of fermentation tank were set at 28 C,pH 6.0 and stirring speed 200 rpm/min.In the fermentation process,after the glycerol supplement,starvation culture and methanol induction,the fermentation broth was purified and freeze-dried by ultrafiltration concentrator.Finally,10 g IFN-? protein product of Takifugu rubrripes was obtained.The same method was used to ferment the interleukin-2 protein products of Takifugu rubrripes by the genetic engineering bacteria LB-IL-2,which had been constructed in the laboratory.The fermentation conditions were 37 C,pH 6.0,and the stirring speed was 200 rpm/min.After induction of fermentation ITPG inducer,the fermentation broth was collected,concentrated by ultrafiltration and purified by nickel column chromatography.Finally,10 g IL-2 protein products were produced by freeze-drying.Through the research of this subject,the step-by-step amplification,separation and purification of recombinant IL-2 and IFN-? produced by fermentation were preliminarily completed,and the purity and molecular weight of the product were analyzed,which laid a foundation for subsequent research and application.