Preparation Of CD4-1 And CD4-2 Monoclonal Antibodies And Analysis Of Positive Cells In Grass Carp

The immune system of vertebrates is an efficient,orderly and systematic system,and immune cells are inseparable from the process of maintaining the body’s homeostasis.CD4~+T helper cells(T help,Th)are an important immune cell population in the immune system and express CD4 receptors on their cell surfaces.In higher animals,the CD4 receptor is a single-chain transmembrane protein encoded by one copy of the gene,and its extracellular contains four Ig-like domains;in fish,two copies of the CD4 gene are found,namely CD4-1 and CD4-2,of which 2-4 Ig-like domains are expressed in the extracellular domain.In fish,CD4-1 and CD4-2 genes have been cloned and identified in a variety of species,and there are many studies on their gene structure and genetic evolution information,but the preparation of their antibodies and the analysis of positive cells are studied.There are really few reports.Grass carp(Ctenopharyngodon idella,Ci)is an important freshwater aquaculture fish in my country.In 2020,the aquaculture production reached 5.7711 million tons,which is the largest species in my country in terms of aquaculture quantity and aquaculture area.However,the increase of breeding density is easy to cause diseases and pest problems.The research on the immune system of grass carp can not only deepen the understanding of the mechanism of fish resistance against diseases,but also lay the foundation for the early laboratory observation of artificial immune drugs.In this study,according to the existing grass carp CD4-1 and CD4-2 gene sequences on NCBI,we constructed the recombinant expression plasmid of the extracellular domain with strong antigenic activity,and used the E.coli prokaryotic expression system to obtain its high-purity protein,and used to immunize mice to produce monoclonal antibodies in vivo.After preliminary screening by ELISA and Western blotting,antibodies with strong specificity to prokaryotic recombinant proteins were obtained,and then the eukaryotic proteins produced by the CHO-S expression system,as well as the identification of confocal microscopy and flow cytometry,showed that the obtained Ci CD4-1 And Ci CD4-2 monoclonal antibody has good specific reaction with the recombinant ectodomain protein expressed in CHO-S cells and the natural CD4 molecule of grass carp.Among them,Ci CD4-1 monoclonal antibody does not recognize Ci CD4-2,and vice versa.In addition,by analyzing the percentage of lymphocytes in immune tissues of healthy fish by flow cytometry,we found the distribution of CD4-1~+and CD4-2~+cells in grass carp.In summary,the results achieved by this study are as follows:1.Using the E.coli expression system,the prokaryotic proteins of grass carp CD4-1 and CD4-2 were simultaneously prepared and purified,with molecular weights of 44 k Da and 37 k Da,respectively.Using prokaryotic recombinant protein and natural membrane protein of grass carp to immunize mice,after screening,two monoclonal antibodies were prepared and purified and named respectively.2.Using the CHO-S eukaryotic expression system,prepare the eukaryotic proteins of CD4-1 and CD4-2,and perform Western blotting analysis on the monoclonal antibodies respectively.The results show that the monoclonal antibodies have good specificity,and CD4-1 and CD4-1 and There is no cross-reactivity with CD4-2antibodies.At the same time,CD4 antibody can recognize the CD4 molecule of grass carp primary head kidney cells,and it is distributed on the cell membrane in a punctate pattern under the confocal fluorescence microscope.3.Using monoclonal antibodies(GC7-CD4-1 and GC7-CD4-2)to perform flow cytometry to analyze the number of CD4-1~+and CD4-2~+cells in each immune tissue of healthy grass carp.The analysis showed that there were significant differences in the proportions of these two types of CD4~+cells.Among them,CD4-1~+cells were 17.6±6.7%,13.1±5.4%and 7.3±4.5%in head kidney,spleen and blood,respectively;The proportion of cells was 22.5±7.8%,18.6±9.9%,8.8±3.8%,and the proportion of the two was the highest in the head kidney,followed by the spleen,and the least in peripheral blood.