PREPARATION OF POLYCLONAL ANTIBODY AGAINST IGM, IGZ AND IGD IN GRASS CARP (CTENOPHARYNGODON IDELLUS) AND EXPRESSION ANALYSIS IN TISSUES

The grass carp (Ctenopharyngodon idellus) is one of the most important freshwater fish in aquaculture industry of China. However, grass carp breeding is plagued by a number of diseases, mostly caused by viruses and bacteria, so the understanding of its immune system is essential for its aquaculture. In this paper, the expression, purification, and polyclonal antibody preparation of the IgM, IgZ and IgD gene were described. The expression primers were designed based on the reported open reading frame (ORF) of IgM, IgZ and IgD gene from grass carp, and incorporated with KpnI and HindIII restriction sites. PCR cycling conditions were as follows: 95°C for 5min; 95°C for 45s, 56°C for 1min, 72°C for 1min for 30 cycles; 72°C for 6min. The IgM, IgZ and IgD gene were cloned into pET-32a expression plasmid at the KpnI and HindIII restriction sites, and the recombinant plasmids were verified by sequencing. The recombinant plasmids were transformated into Escherichia coli Rosetta-gami (DE3) strain. Sodium dodecyl sulfate- polyacrylamide gel elactrophoresis (SDS-PAGE) analysis showed that recombinant protein were satisfactorily expressed by optimizing the concentration and induction time of Isopropyl_β-D-1-thiogalactopyranoside (IPTG). All recombinant proteins were expressed as inclusion bodies in agreement with the expected molecular weight, and were purified successfully by using affinity chromatography in the denatured condition. The purified proteins were then injected into Japanese white rabbits in order to obtain polyclonal antisera. Western blot analysis showed that the purified IgM, IgZ and IgD recombinant proteins were recognized by the rabbit antisera, respectively. By using the rabbit antisera of IgM and IgZ, Western blotting showed that IgM and IgZ expressed in intestine, head kidney, trunk kidney, skin, spleen, brain, gill and blood.