Preparation Of Polyclonal Antibodies And Analyses Of Expression Of Zebrafish Myh6 And Nodal Genes And Drosophila CG3295 Gene

Heart development is an extremely complicated procedure. Zebrafish model is one of the ideal models which are used to study the molecular mechanisms of cardiac development. Myh6 is a marker gene in cardiac development which promote expansion of ventricular myocardial cells. Nodal encodes transcription factors which can regulate left-right axis asymmetry of heart. At present, there are few reports in which antibodies were used for the protein expression and functional studies in zebrafish model. Drosophila CG3295 gene (homologous vertebrate genes are RMND5A/B) is a new candidate gene involved in heart development that was found by our lab, and its function remains to be clarified. This studies mainly focus on the preparation of polyclonal antibodies and analyses of expression of zebrafish Myh6 and Nodal genes and Drosophila CG3295 gene are performed.To obtain the expression plasmids of the Zebrafish Myh6 and nodal, Zebrafish total RNA was isolated and its cDNA was obtained by reserve transcriptional PCR. The 440bp fragment of Myh6 was obtained by PCR amplification, and then was cloned into pGEX-4T-1 vector and identified by sequencing. The recombinant expression plasmid containing Myh6 gene was transformed into BL21 and the fusion protein was induced by IPTG. The purified protein obtained by immobilized Glutathione Sepharose was immuned the New Zealand white rabbits to prepare antibody. Western blotting showed that the antibody titer was 1:1000. The assay of Myh6 expression with zebrafish tissues using Myh6 polyclonal antibody, revealed that the Myh6 protein was expressed in heart and muscle. Zebrafish embryos immunohistochemical analysis showed that the Myh6 protein signal can be detected in heart tissue.Using Zebrafish cDNA as the template, the 309bp of Nodal fragment was obtained by PCR amplification, and then was cloned into pET-28a vector that was identified by sequencing. The recombinant expression plasmid containing Nodal fragment was transformed into Rosseta and the fusion protein was induced by IPTG. The protein was purified by Ni-IDA affinity gel column. The purfied protein was used to immune the New Zealand white rabbits to prepare antibody. Western blotting showed that the antibody titer was 1:1000, and the Nodal protein was expressed in several Zebrafish tissues including heart, and its protein expression began at multi-cell stage in embryo.For obtaining the expression plasmid of the Drosophila CG3295 gene, the Drosophila cDNA was used as the template. The 540bp fragment of CG3295 was obtained by PCR amplification, and then was cloned into pET-28a vector and identified by sequencing. The recombinant expression plasmid was transformed into Rosseta and the fusion protein was induced by IPTG. The protein was purified by Ni-IDA affinity gel column. The protein purified was used to immune the New Zealand white rabbits to prepare antibody. Western blotting showed that the antibody titer was 1:1000, and that showed significant changes of CG3295 protein expression were detected in different stage of Drosophila embryo, however, the expression level of CG3295 was stable at the phases from the formation of the embryonic heart precursor cells to the differentiation of heart cells. Immunohistochemical analysis of Drosophila embryos showed that the CG3295 protein was expressed in the heart and other tissues.The results showed that the prepared Myh6, Nodal and CG3295 polyclonal antibody titer were high, and they could be used to study these genes protein expression in Drosophila and zebrafish by the embryo immunohistochemical, respectively. These lay a solid foundation for our further studies of the function of Myh6, Nodal and CG3295 gene.