Transcriptome Analysis Of The Gill Mucosa Of Lampetra Japonica And Preparation VLRC Polyclonal Antibody

Mucosal immune system is an important component of animal immune system.Lamprey lives in water,and their skin and gills are in direct contact with the water environment,so the gill mucosa is an important defense tissue against infection.In order to explore the genetic basis of the immune response of lamprey gill mucosa,the second generation RNA-Seq technology is used to sequence the gill tissue of lamprey.The differential expression of immune related molecules between mixed-bacteria stimulated group and control group lampreys was analyzed by bioinformatics method.The results showed that: 1.After stimulation by mixed-bacteria,the relative expression levels of some molecules related to physiological processes in the gill mucosa are down-regulated.These molecules include:myosin light chain family(MYL),myosin heavy chain family(MYH),calcium myosin T family(Troponin T,TNNT),etc.,indicating that the organism exhibits physiology reduced activity after infection with pathogenic bacteria.2.The relative expression level of signaling molecules related to TCR signaling pathway of higher vertebrates is up-regulated.These molecules include: mitogen activated protein kinase p38(p38MAPK),NCK adaptor protein 1(NCK),and tyrosine protein kinases Lck,Fyn,and Tec.These indicate the lamprey gill mucosa has the immune response of the immune system which has a molecular basis that is conserved with the TCR signaling pathway in higher vertebrates.3.The number and types of VLRA and VLRC receptors expressed by T-like cells in the lamprey gill mucosal system are dominant.However,the expression level of germline VLRC has a clear upward trend,and functional VLRC has also occurred from scratch.The changes in rearrangement indicate that the lamprey gill mucosal immune system mainly rely on VLRA and VLRC,two T cell receptor-like immune responses.In order to further study the role of VLRC in the immune response of lamprey,this paper prepared a rabbit polyclonal antibody of this molecule,which laid the foundation for further research on its function in the future.By homologous alignment of multiple VLRC protein sequences,it was found that the highly conserved domains are LLRCT and LRRNT,and the sequence identity is 85% And 63%.The online website predicts the epitope of the VLRC molecule.It is found that among the 13 epitopes detected,the peptides located at the 38 th to51st amino acids of the LRRNT domain have better antigenicity,and the LRRNT domain is determined to be Antigen peptides.Then,the expression vector was constructed from the LRRNT sequence to induce expression,and it was found that the expression induction effect was best when the condition was 37?and the IPTG is 1 mmol/m L.Purify the induced protein,mix the purified soluble protein LRRNT with an adjuvant to make an antigen,and subcutaneously immunize rabbits(Oryctolagus cuniculus).After 4 boosting immunizations,the final titer is as high as 1:128000 polyclonal antibodies.The purified antibody is detected by immunoblotting,and it was found that the prepared polyclonal antibody could not only recognize the recombinant protein but also detect the native VLRC receptor protein in the sample prepared by the gill tissue.The above results indicate that the VLRC polyclonal antibody prepared in this paper has a high specificity,which can provide a guarantee for the subsequent smooth development of VLRC functional research.