Preliminary Study On The Diversity Of Immunoglobulin V Region And The Function Of Aicda Gene Of Cyprinus Carpio L.

Immunoglobulin(Ig)is the most important component of adaptive immune system.The diversity of variable regions of immunoglobulins comes from a variety of mechanisms,including germline gene recombination and somatic high mutation.In the process of immunoglobulin production,recombination activation gene(Rag)and activation induced cytosine deaminase(AID)are the key factors.Rag rearranges the V(D)J gene scattered on the Ig locus to form the variable region of immunoglobulin;AID is encoded by Aicda gene,which can lead to the deamination of cytosine nucleotides in DNA and the formation of uracil,resulting in G:U mismatch.In the subsequent mismatch repair process of mammals,it can further trigger somatic high mutation(SHM),gene conversion(GC)and class conversion(CSR),thus greatly enriching the diversity of immunoglobulins.The research on antibody diversity and production mechanism of bony fish is limited,especially the function of AID in fish antibody production has not been studied.In this study,Cyprinus carpio L.,a common economic fish in China,was used as experimental materials.58 Ig M variable region sequences and 16 Ig Z3 variable region sequences were cloned by 5’RACE method.The international immunogenetic database(IMGT)was used to analyze the variable region sequences from Ig M and Ig Z3,and the sequence alignment was carried out in the common carp Ensembl genome database to obtain the chromosome localization of each V region gene f Ragment of common carp.Using Ig-blast tool for sequence analysis,it was found that the V-region sequence belonged to V3 gene family.In the obtained sequence,exceptζ7 sequence alignment to the human V1 family,the other sequences have high homology with the human V3 family,indicating that the human V3 family is relatively old in evolution,and the preference of Ig M and Ig Z3 to use V3 subfamily is different.This study also found the phenomenon of stop codon in CDR3 for the first time,which may be caused by high somatic mutation.At the same time,no N/P nucleotide insertion was found in Ig M,which also led to the shortening of CDR3.After further comparison,it was found that there were at most two nucleotide differences between the expressed V gene and the embryonic V gene,suggesting that common carp may have somatic high mutation effect.Secondly,the expression of Rag1,2 and Aicda genes in the early development of common carp was detected by fluorescence Real-Time PCR.The results showed that the expression of Rag1 was very low in the first four days after fertilization,almost no expression was detected,and reached the peak on the 24th day;The expression of Rag2 was low on the 2nd-10th day after fertilization,and reached the peak on the 16th day after fertilization;Aicda was expressed at a low level 1-4 days after fertilization,reached the highest level on the 6th day and the lowest level on the 24th day.Rag is involved in the rearrangement of TCR and BCR germline genes,while AID has the function of cytidine deaminase.The above results show that Rag and AID have the function in the early development stage of common carp.In addition,the Ti and Td antigen stimulation model was constructed by 6-month-old carp,and the expression and localization of Rag1,Rag2 and Aicda genes in spleen,head kidney and isolated spleen lymphocytes were detected.The results showed that Ti and Td antigens could induce the expression of Rag in spleen and Aicda in head and kidney.The expression of Aicda could be detected in spleen lymphocytes after immune stimulation,and reached the peak on the third day after stimulation.The results of immune stimulation experiment showed that both Ti and Td antigens could activate antigen-dependent Rag recombination activity.At the same time,the expression of Aicda was not only located in the head,kidney and spleen,but also expressed in lymphocytes.In addition,in situ hybridization of spleen tissue further showed the expression of Rag and Aicda.These results laid a foundation for further study on the function of Rag and AID in antibody production in common carp B cells.Thirdly,the Aicda gene of common carp was cloned for the first time,p ET22b-AID expression vector was constructed,Rosetta was transformed,the induced protein was purified,and AID deaminase activity was detected in vitro.The results showed that the expressed and purified AID had deaminase activity,which could deamination deoxycytidine,cytosine nucleoside and cytarabine.The AID antibody was obtained by screening the AID protein expressed in prokaryotic cells,and the AID and Ig Z3 antibodies were detected by immunofluorescence.The results showed that there was the expression of AID in Ig Z3~+B cells.This experiment also amplified the promoter sequence of common carp Aicda gene.Sequence analysis showed that the promoter sequence of common carp Aicda was 76.4%similar to zebrafish and low similar to mammals.Further analysis showed that the promoter sequence could be combined with immune related transcription factors such as sp1,hdac3 and sp2,which provided a basis for further study on the expression regulation of common carp Aicda gene.