Molecular Cloning And Function Analysis Of Transcription Factor Blimp-1 In Common Carp (Cyprinus Carp L)

B lymphocyte induced mature protein 1(Blimp-1)is an important transcription factor involved in B cell differentiation in vertebrates.Different stages of B lymphocyte differentiation are regulated by different transcription factors.Among them,in the process of B cell differentiation,Blimp-1 is expressed in plasma cells,and B cells in germinal center.Blimp-1 is involved in the regulation of the development of many types of tissues and regulates the process of cell differentiation.In addition,higher levels of ?-isoform expression of Blimp-1 can also be detected in the malignant transformation of normal plasma cells.Blimp-1 is a member of the PR gene family,which is characterized by an unequal number of ZnF-C2H2 domains.The DNA-binding domain of this gene is highly conserved in vertebrates and the homology between zebrafish,Xenopus laevis,Takifugu rubripes and mouse exceeds 93%.There is a few research about blimp-1 in common carp.In this study,the blimp-1 gene sequence was cloned by analyzing the conserved regions of blimp-1 sequence in different species,and the sequence characteristics were analyzed and the evolutionary tree was constructed.In this experiment,the squid model of immune agitation by Aeromonas hydrophila soaked and injected was constructed.At the same time,tissue materials from 8 different developmental stages from the first day after fertilization to 21 days of incubation were obtained.The expression of blimp-1 gene at the mRNA level in the above tissues was detected by PCR.In addition,a blimp-1 prokaryotic expression vector was constructed,and a partial fragment was induced and purified.Polyclonal antibodies were prepared and the expression of Blimp-1 in different developmental stages was detected.The cDNA of blimp-1 was 2765 bp,and the ORF was 2334 bp,which encoded 778 amino acids.Structural analysis showed that the gene has a SET domain and five ZnF-C2H2 domains.Real-time PCR results showed that blimp-1 was expressed in all tissues.Among them,expression was highest in the intestine,followed by spleen,head kidney and liver.The expression of blimp-1 in the intestinal tract was highest after real-time PCR was used to detect the tissue immersed with Aeromonas hydrophila and injected with immunostimulation.Among them,expression was down-regulated in the spleen tissue of the injection group,while the more pronounced up-regulation of the immersion stimulation group appeared in the midgut tissue.In developmental fish,the test results showed that the expression of blimp-1 could be detected in fertilized eggs on the first day after fertilization,while the expression levels were lower in the eight different developmental stages after fertilization.The above experimental results preliminarily showed that blimp-1 was expressed in each immune-related tissue,suggesting that it plays a role in the B cell response process of the above-mentioned tissues.In both stimuli,changes in response to different tissues indicate that both B-cell responses in systemic immunity and B-cell responses in mucosa-associated lymphoid tissues are likely to be involved in blimp-1 and have different patterns of response.Western blotting analysis of Blimp-1 expression at developmental stage showed that Blimp-1 expression was less during fertilized eggs and highest during ovulation.Contrary to the expression level of blimp-1 at the mRNA level,blimp-1 mRNA was expressed at the highest level in fertilized eggs and decreased at the time of ovulation.It is possible that the blimp-1 mRNA is carried in the matrix and expressed during the ovary of the eye.The Blimp-1 protein is involved in the differentiation and development of various tissues of the embryo.Carp is the primitive vertebrate that first involve in acquired immunity.The study of its B lymphocytes and their ability to secrete antibodies will benefit to more understanding of the evolution and mechanisms of vetebrate acquired immunity.Blimp-1 as a transcription factor plays an important role in embryonic development and B cell differentiation.In this experiment,the gene cloning and quantification of its expression and the preparation of polyclonal antibodies will be the basis of further functional research.