Preparation Of Polyclonal Antibody And Analysis Special Expression Of Three Genes Of Zebrafish Heart Development Genes And Drosophila Gene CG8419

Cardiogenesis is a very complex process and it requires precise regulation of series of genes. The molecular mechanism underlying heart development is complex, involving in the exact spatio-temporal expressions of a series of heart genes. Heart development is regulated by a series of transcription factors. The mutations and abnormally spatio-temporal expressions of these genes will result in mortality in pregnancies and cardiac disease. It is a critical prerequisite to understand the normal progress of cardiovascular development and the mechanisms underlying the causes of cardiac diseases.vmhc is a marker gene and its expression is restricted to the cardiac ventricle in zebrafish. Lefty-1 and charon (the ortholog of cerl-2 in mouse) plays an important role in establishment of correct L/R asymmetry. zebrafish is proved to be an ideal animal model to study the mechanisms underlying cardiovascular development. At present, there are few reports in international jounals to us the antibodies of lefty-1 and charon to research the protein expression and gene function in zebrafish model. A heart developmental candidate gene CG8419 (the ortholog of TRIM45 in vertebrates) was cloned by our lab, and the primary results showed that the mRNA levels of key hematopoietic gene Srp, Lz and Gcm were significantly downregulated in the mutation larva of the CG8419 gene, suggesting that CG8419 may be involved in Drosophila hematopoietic development. So, generating high quality rabbit serum and mouse serum of these genes is usful to further study the mechanisms underlying cardiovascular development.Here, the hydrophilic antigen regions of the Zebrafish gene vmhc, lefty 1, Charon and Drosophila gene CG8419 selected by biological information analysis were obtained by PCR amplification, and then were inserted respectively into two kinds of vectors to generate the prokaryotic expressing system. The recombinant plasmids were transformed to E.coil. Expression of the target proteins were induced by IPTG and assayed by SDS-PAGE, respectively. After purified, the fused proteins were injected into New Zealand big white rabbits or balb/c mouse to produce polyclonal antibody, respectively.The titer of the vmhc antibody identified by Western blot was 1:3000. The heart tissue distribution of vmhc was detected by immunohistochemistry indicated that vmhc expresses in nuclear. The titer of the vmhc mouse polyclonal antibody was greater than 1:3000. Zebrafish embryos Staining showed that vmhc expresses in heart ventricle.The specificity of Lefty 1 antibody was determined by Western blotting and the results indicated that the titer was 1:2000. Analysis of lefty 1 expression distribution in tissue protein show that lefty 1 expresses in the early development stage of zebrafish.The titer of the charon antibody tested by Western blot was 1:2000. Western blot with the tissue protein suggested that charon expresses strongly in cardiac tissue, and the express slightly in gut and skin. The antibody staining of zebrafish suggested that Charon expresses mainly in heart field.The analysis results showed that polyclonal antibody CG8419 is high in specificity and sensitivity and the titer was about 1:2000. Western blot of the tissue protein suggested that CG8419 expresses in larval stage and the antibody staining of Drosophila embryos indicated that CG8419 expresses in salivary gland and other organs.These results showed that each polyclonal antibody was of high sensitivity and specificity that may be used for further analysis of these gene’s expressions in embryonic development. These antibodies will facilitate further our studies on the molecular mechanisms of heart development and function of these genes in zebrafish.