Preparation Of Polyclonal Antibody And Expression Of Four Heart Development Genes In Zebrafish

The genome of the animal model zebrafish includes 30000 genes, which has 87% homologization compared with human’s. The blood circulation of zebrafish is single circulation, the heart includes one venous sinus, one ventricle and one auricle. Compared with human’s, zebrafish has many similar characteristics in heart-blood vessel, blood, alimentary tract, liver, kidney and visual system. Zebrafish is easy to raise, the embryo is large and transparent completely, easy to observe, the rate of development is fast and the phenotype is identited, so it is an ideal model in researching heart development.The pitx2 gene mediates the left-right asymmetry development of heart and laterality of internal organs, which is the last one of target gene in left-right signal pathway.Myosin light chain regulatory factor myl7 is a marker gene of heart development. Myl7 is involved in sarcomeric hypertrophic cardiomyopathies in humans. Wntll is a key gene of non-classic Wnt signal pathway that regulates the heart development.Klhl31 participates the development process of heart, and its function remains to be clarified. Until now, the protein expression and the function research of the genes by using antibodies are rarely reported in zebrafish.The parcial coding region of the pitx2、klhl31、wnt11 and myl7 genes were amplified by PCR and then was cloned into pGEX-4T-1 vector. After confirmed by sequencing, the recombinant espression plasmids, were transformed into BL21 cells and the expressions of fusion proteins were induced by IPTG. The purified proteins were affinity purificated by GST beads, and then immuned the New Zealand white rabbits to prepare antibodies. Another my17 fusion protein immuned Mus musculus albus. Finally 5 kinds of polyclonal antibodies were obtained. Using Western Blot, the antibody titer and specification was identified. Generation rabbit serums of anti-Pitx2、anti-Klhl3、anti-My17、anti-Wnt11, antibody titer were as following:1:2000,1:2000,1:1500,1:2500. The antibody titer of mouse serums anti-my17 is 1:3000. We used zebrafish tissues and organs to extract proteins to detect characters of pitx2 and klh131 antibody. The result showed that the protein of pitx2 was expressed in heart, brain, muscle, intestine and liver, specially in heart with a higher level. The klh131 protein was expression in heart, bowels, eyes, ovaries, with a higher level in heart. Analysis of zebrafish embryo antibody staining indicated the pitx2 protein and the myl7 protein.The pitx2 protein was expressed in 15 somites in left-right asymmetry.We can observe the location of the cardiae muscle progenitor cells perspicuously,in 22 somites.Moreover expression matter of pitx2 and my17 in heart of zebrafish 48hpf can be observed.We ues anti-my17 of mouse serums in this exprement.The results above suggest that the 5 polyclonal antibodies can be used to research the protein expression in zebrsfish embryo and organs, which provide an effective research method for our research work on heart development.