Antibody Production And Related Immunological Studies Of The VLRA In Lamprey

There are two major groups of vertebrates and invertebrates in the animal kingdom.Not only the adaptive immune system but also the innate immune system exists in vertebrates;in invertebrates only the innate immune system exists.Among the higher vertebrates in vertebrates,its adaptive immune system is divided into T-cell-mediated cellular immunity and B-cell-mediated humoral immunity.Among the jawless vertebrates,there are no T cell receptors(TCR),B cell receptors(BCR)and major histocompatibility complexes(MHC)in higher vertebrates,but it also has an adaptive immune system through a novel variable lymph An adaptive immune system formed by cellular receptors.At present,there are three types of VLR,namely VLRA,VLRB and VLRC,which exist on three different lymphocyte subsets.VLRA and VLRC are equivalent to T cells in the adaptive immune system of jawless vertebrates,while VLRB is equivalent to B cells.Lamprey(Lampetra japonica),as a model organism in jawless vertebrates,has high research value.In this study,antibody preparation and functional studies on VLRA molecules were carried out,in order to further understand the adaptive immune system of lampreys.The molecular response mechanism laid the foundation.In this paper,through the amino acid sequence alignment of several typical VLRA molecules,the LRRNT,LRR1,LRR variable regions,LRRCT,CP and 3'-terminus domains were found,because the LRRNT domain and the LRRCT domain are conserved High,the similarity rate is 61% and 60% respectively,so the epitope prediction is performed.The results show that the antigen immunogenicity of the LRRNT and LRRCT are both strong,because VLR is an anchoring protein Glycosylphosphatidylinositol(GPI)is connected to the surface of lymphocytes,so the LRRNT domain is more suitable as an antigen for the free end.After designing primers with LRRNT ends of the restriction site,the c DNA sequence of this domain was amplified by nested PCR method,with a total of 117 bp,encoding a protein containing 39 amino acid residues.The target fragment of VLRA antigen was ligated with p ET-32a(+)expression vector,and then the prokaryotic expression vector of VLRA-p ET-32a(+)was successfully constructed.Transform the recombinant plasmid into E.coli Rosetta competent cells to induce expression,,the final IPTG concentration was 0.1mmol / L,and induction at 28 ? for 2h was the best condition.The protein was purified by Ni column affinity chromatography.The purified recombinant protein was used as an antigen to immunize adult New Zealand rabbits(Oryctolagus cuniculus).One week after the fourthbooster immunization,the results of enzyme linked immunosorbent assay(ELISA)showed,The antibody titer has reached 1: 128000,the New Zealand rabbits were subjected to ear artery blood sampling,antiserum preparation and antibody purification.Western blot results show that the antibody has strong specificity for VLRA recombinant protein and VLRA natural protein in gill tissue.We are in the peripheral blood mononuclear leukocytes,gill tissues and myeloid bodies of lamprey,Western blot experiments were used to detect the protein expression profile of VLRA in various tissues.The results showed that no matter whether it was a control group or an immunized group,the specific band of VLRA was detected only in the gill tissue,and after 36 hours of immunization with PHA The relative expression in gill tissue was 2.48 times that of the control group,reaching a very significant difference.The results of Co-Immunoprecipitation(Co-IP)experiments showed that there was an obvious difference band at about 60 k D,which presumably interacted with the VLRA protein in the natural protein.The subsequent results will be verified by mass spectrometry.The above results indicate that VLRA participates in PHA mediated immune response in Lamprey's adaptive immune system,laying a foundation for the subsequent exploration of the role of VLRA variable lymphocyte receptors.