Preparation Of Antibody Against Lja-VLRB Molecule Of Japanese Lamprey And Related Immunological Studies

The lamprey belong to Chordata,Cyclostomata,Petromyzoniformes.It is a representative of invertebrate vertebrates.It is located between invertebrates and vertebrates and has a unique evolutionary position.Because of the adaptive immune defense system mediated by VLR,it has attracted wide attention from immunologists.In this study,bioinformatics was used to analyze the VLRB open reading frame c DNA(Lja-VLRB)of Lampetra japonica.The analysis results showed that the VLRB open reading frame was 822 bp in length,encoding a total of 274 amino acids and divided into 6 domains.They are SP,LRRNT,LRR1,LRRV1,LRRV2,LRRCP,LRRNT.The calculation of the conservativeness of each domain of the nucleic acid and amino acid sequence shows that the LRRNT,LRR1 and LRRCT domains are the most conserved,at 78%.Antigen epitope software predicts that LRRNT,LRR1 and LRRCT all have antigen-binding epitopes.Since the LRCCT end anchored on the cell membrane in the form of GPI binding is not conducive to antigen binding,the LRRNT and LRR1 domain sequences were finally selected as the antigen sequence for preparing anti-VLRB antibodies.Primer5.0 software was used to design the amplification primers containing the restriction site and the protection base of the restriction site,and the N-terminal antigen sequence of the lamprey VLRB molecule was successfully cloned.The nested high-fidelity enzyme PCR amplification technology was used to connect the N-terminal antigen sequence of the Lja-VLRB molecule to p ET-32a(+)to construct the Pet-32a-Lja-VLRB recombinant plasmid.The recombinant plasmid was introduced into E.Coli Rosetta expressing bacteria to induce protein expression and purification.After mass spectrometry detection,the purified Lja-VLRB protein was used as an antigen to boost immunization of Oryctolagus cuniculus multiple times to prepare polyclonal antibodies.Anti-serum was isolated and purified by blood sampling from the ear artery.The antibody titer detected by ELISA reached 1: 1024000.After purification,the polyclonal antibody was tested by immunoblotting using anti-His tag antibody and showed that it had strong specificity for both recombinant protein and natural protein.Using mixed bacteria,LPS,PHA,immunostimulation lamprey,killed at 24 h.Western blotting experiments were used to detect the protein expression of Lja-VLRB in various tissues before and after immunostimulation.The results showed that Lja-VLRB was expressed in leukocytes,myeloid bodies,and gill tissues before immunization,and the expression level of leukocytes was highFor the other two organizations.After immunization with mixed bacteria,the expression of Lja-VLRB protein in myeloid body was significantly up-regulated(P <0.05)about 2 times that of the blank control,and the expression of Lja-VLRB protein in white blood cells was significantly up-regulated(P<0.05),which was about the blank control 1.5 times,the gill tissue expression level is almost unchanged,the results show that Lja-VLRB plays an immune role in B-like lymphocytes against the invasion of foreign pathogens.After LPS immunization,the expression of Lja-VLRB protein in white blood cells showed a very significant upregulation(P<0.01),which was about four times that of that without immunization.The expression of Lja-VLRB protein in gill tissues and myeloid bodies was significantly upregulated(P<0.05)About twice as high as when not immunized.LPS can stimulate the proliferation and differentiation of B lymphocytes and enhance the antigen presentation ability.The results further prove that VLRB is similar to B lymphocytes,and Lja-VLRB is widely expressed in peripheral blood and mainly plays an important immune role in lymphocytes.Myeloid body is the main place for VLRB + to mature,so the expression level in myeloid body will increase significantly after invasion by foreign pathogens.Gill tissue is the main place for VLRA +,and the content of VLRB + is less.The expression of Lja-VLRB is stimulated by LPS Significantly increased the amount may indicate that VLRB and VLRA have a synergistic effect in exercising immune function.After PHA immunization,the expression of Lja-VLRB protein in myeloid body was significantly up-regulated(P <0.05),which was about 2.5 times that of the blank control,and the expression level of leukocytes was significantly up-regulated(P<0.05),which was twice that of the blank control.There was no significant change in expression level in tissues(P>0.05).Previous studies have shown that PHA induces T cell activation and proliferation by binding to mitogen receptors on the surface of T cells.After being stimulated by PHA,VLRA,which is similar to T lymphocytes,should have an immune effect.Studies have shown that the expression of VLRA in gills of lamprey is significantly increased after PHA immunization.However,the results of this study show that the expression of Lja-VLRB in white blood cells and myeloid bodies is upregulated after PHA immunization,and the expression in gill tissues is basically unchanged,indicating that VLRB and VLRA participate in immune activities in different tissues and may have a synergistic effect.We preliminary studied the role of VLRB molecules in the immune response process,which laid the foundation for further research on the function of VLRB molecules and the preparation of new alternative antibodies.