Molecular Cloning And Developmental Analysis Of Zebrafish Cd34 Family And The Production Of Cd34 Polyclonal Antiserum

This thesis focuses on the mining and expression during embryo development of zebrafish homologs of mammalian cd34 family genes,as well as the production of anti-serum against zebrafish CD34L1 and CD34L2.In a preliminary in NCBI database and Ensembl database,predicted zebrafish podxl was found,while zebrafish cd34 and podxl2 could not been found.By a further search with gene synteny analysis and blast,we obtained two zebrafish orthologous genes of cd34(si:ch211-286o17.1 and si:dkey-261h17.1)in the Ensembl database and and podxl2 homologous gene(ZGC: 171566).si:ch211-286o17.1 and si:dkey-261h17.1 were named as cd34l1 and cd34l2,respectively.The expression of cd34l1 and cd34l2 in zebrafish development were unknown yet.Therefore,we first used the RACE method to clone the cDNA sequences of cd34l1,cd34l2,podxl and podxl2 and run the bioinformatical analysis.The expression of cd34 family genes during zebrafish embryo development was further detected by semi-quantitative RT-PCR.Moreover,the whole mount in situ hybridization was carried out to characterize the expression pattern of cd34l1 and cd34l2 during zebrafish embryo development.Furthermore,by constructing CD34L1,CD34L2 and CD34L2-2 prokaryotic expression recombinant plasmids,CD34L1,CD34L2 and CD34L2-2 recombinant proteins were expressed and purified,and subcutaneously injected into rabbits to immunize experimental rabbits to prepare polyclonal antisera.Indirect ELISA was run to determine the titers of antiserum and Western blot was run to determine the specificity of these antiserum.The results show that: 1.Cloning and bioinformatic analysis of cd34 family gene cDNA in zebrafishThe cDNA of cd34l1 was 1631 bp,and ORF length was 867 bp,encoding 288 amino acids.The cDNA of cd34l2 was 1975(another splice cd34l2-2,1918 bp)bp,and ORF length was 828 bp(771 bp),encoding 275(256)amino acids.The cDNA of podxl was 2843 bp,and ORF length was 1338 bp,encoding 445 amino acids.The cDNA of podxl2 was 2457 bp,and ORF length was 1764 bp,encoding 587 amino acids.Analysis of the protein structure showed that the zebrafish CD34L1,CD34L2,CD34L2-2,PODXL and PODXL2 proteins are transmembrane proteins,and each includes a signal peptide,a transmembrane region,and a Pfam: CD34_antigen.Phylogenetic analysis showed that the zebrafish CD34L1,CD34L2,CD34L2-2,PODXL and PODXL2 clustered with fish and then with mammals.2.The expression analysis of cd34 family genes during zebrafish embryo developmentSemi-quantitative RT-PCR studies of zebrafish cd34l1,cd34l2,cd34l2-2,podxl and podxl2 expression during zebrafish embryo development.At 6 hpf the expression of zebrafish cd34l1 and podxl could not be found,while the expression of zebrafish cd34l2 and podxl2 were found.In all the selected time points,zebrafish cd34l2-2 was not expressed.Subsequently,the p GEMTcd34l1-A and p GEMT-cd34l2-A plasmids were constructed for antisense probe synthesis,and the expression of cd34l1 and cd34l2 genes during zebrafish embryos development was detected by whole mount in situ hybridization.The results showed that at 12 hpf,cd34l1 and cd34l2 were concentratedly expressed at the embryonic circle,and then gradually expressed around the thymus,kidney and eyes.3.Production and verification polyclonal antiserum of zebrafish CD34L1,CD34L2 and CD34L2-2Recombinant prokaryotic expression plasmids of extracellular specific fragments of zebrafish CD34L1,CD34L2 and CD34L2-2 were constructed and transformed into E.coli BL21(DE3)strain to induce expression.The recombinant protein was purified by using Ni sepharose 6FF affinity beads,and subcutaneously injected into rabbit in combination with adjuvants(FCA/FIA)for four times.Indirect ELISA method was used to test theantiserum titers.It was found that female and male rabbit zCD34L1 and zCD34L2 polyclonal antisera titers were higher than 1: 5120000,that female rabbit zCD34L2-2 polyclonal antisera titers was higher than 1: 5120000,and that male rabbit zCD34L2-2 polyclonal antisera titers was higher than 1: 320000.Western blot results further confirmed that the antisera can not only recognize the prokaryotic fusion protein,but also the recombinant protein expressed by 293 T cells.conclusions: 1.The cDNA sequences obtained by the RACE technology are the zebrafish cd34l1,cd34l2(cd34l2-2),podxl and podxl2 gene cDNA sequences.Bioinformatic analysis shows that zebrafish cd34l1,cd34l2(cd34l2-2),podxl and podxl2 are mammalian counterparts.The presence of cd34l1 and cd34l2 in zebrafish may be due to the unique genome duplication of teleost fish.The existence of cd34l2(cd34l2-2)is likely to be the result of m RNA alternative splicing.2.Expression of cd34l1,cd34l2,podxl and podxl2 during zebrafish early development.Zebrafish cd34l1 and cd34l2 are not only expressed in hematopoietic tissues such as the ICM region、CHT and kidney,but also in non-hematopoietic tissues such as the eyes,brain,and thymus,laying a foundation for further study of zebrafish CD34 family molecules.3.The rabbit-derived zCD34L1,zCD34L2 and zCD34L2-2 polyclonal antisera were successfully produced,which provided a good basic tool for the in-depth study of zebrafish CD34 molecules.