| Complement receptor 2(CR2;CD21)is a surface-associated glycoprotein that mainly expressed on mature B cells.CR2 links the innate immune response and adaptive immune response by binding to C3d that is covalently attached to targets,and which results in a cell signaling phenomenon that lowers the threshold for B cell activation.CR2 plays an important role in the immune system,however,it is needs to be solved that Chicken CR2(ChCR2)gene has not been annotated in National Center for Biotechnology Information(NCBI).So,RNA-seq technology has been used to explore novel genes that is in the process of undermine the mechanism of Marek’s disease vaccine to enhance the immune functionality in Chicken.It was found that one of the novel genes(ENSGALG0000003007)has more than 80%homology with other birds that through sequence alignment analysis.By comparing the gene with Gen Bank database,it was found that the sequence of the novel gene was consistent with that of the annotated AJ720954.1 sequence.Therefore,according to the sequence,specific primers were designed for RT-PCR experiment,a specific band of 1113 bp was amplified,and the sequencing results were consistent with the sequence.First of all,in this thesis,a bioinformatics analysis was performed on the ChCR2gene.The results show that ChCR2 is closely related to CR2 molecules of some birds,and the phylogenetic tree branches are far away from mammals such as human,cattle and other mammals;at the same time,ChCR2 molecule has typical SCR/CCP domains,transmembrane region and cytoplasmic region of CR2;in addition,3D modeling results are also constructed by members of the CR2 family as templates;finally,the results of domains amino acid sequence alignment also show that there are similarities and differences between ChCR2 and Human CR2.In order to further prove that the novel gene is ChCR2,in this study,the interaction and action region of ChCR2 and its ligand ChC3d were identified by pull-down,Co-IP and laser confocal technology,and the expression of ChCR2 in different tissues of Chicken was detected by RT-q PCR.The results showed that ChCR2 and ChC3d could interact with each other,and not only does ChCR2 and ChC3d binding region were determined in SCR1-4 region but also its cellular localization was identical.Surprisingly,ChCR2 is mainly presented in immune organs such as the spleen,bursa and thymus,and also presented in peripheral blood lymphocytes,while almost no expression is found in non-immune organs such as liver and brain.The results were consistent with the expectation.The above experiments also basically confirmed that this novel gene is ChCR2.In order to further verify the distribution of ChCR2 in different immune tissues or immune cells,monoclonal antibody(m Ab)against ChCR2 molecule need to be prepared.First,we have established a monoclonal hybrid cell line secreting mouse m Ab that was prepared by a traditional hybridoma preparation method;then,the cell line was injected into rats to produce ascites and the m Ab was purified;finally,the purified m Ab was identified and its specificity has also been proven.The results showed that hybridoma cell line were successfully obtained,named it 3E9,and the ascites was prepared,14 mg mouse anti-ChCR2 Ig G(m Ab 3E9)was purified;the efficacy of the m Ab 3E9(1.44 mg/m L)was found to be with a titer of 1:2.048×10~6.The results of IFA,WB and antigen cross-reaction showed that the ChCR2 protein could be specifically recognized by m Ab 3E9,and its affinity constant reached 2.82×10~9.Mab3E9 belong to Ig G2b,the light chain is Kappa type,and the recognized epitope is in the250aa-280aa region of ChCR2.In a nutshell,ChCR2 gene has been preliminarily identified,the interaction between ChCR2 and ChC3d was also confirmed,and the binding region of the two were explored at the same time.The successful preparation of ChCR2 monoclonal antibody also provides a powerful tool for subsequent research.The results of this thesis enriched the immunological content of Chicken and even Bird. |
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