Cloning and expression of the constant region of rainbow trout (Onchorhynchus mykiss) μ immunoglobulin chain in Escherichia coli

摘要

The importance of rainbow trout ( Onchorhynchus mykiss ) in Iran aquaculture industry on one hand, and increasing the mortality of this fish due to outbreaks of infectious diseases, on the other hand, indicate the requirement for more profound understanding the rainbow trout immune system and access to laboratory tools for definitive diagnosis of its diseases. One of the most important defense mechanisms of vertebrates including fish is the production of immunoglobulin against microbial pathogens. In rainbow trout, dominant immunoglobulin in serum is immunoglobulin M (IgM). The purpose of this study was the cloning and expression of the constant region of rainbow trout IgM heavy chain (μ chain) gene in Escherichia coli . Therefore, RNA of the targeted gene was extracted from spleen and head kidney of rainbow trout and the constant region of μ chain was amplified by RT-PCR. The amplified fragment was ligated to pMALc2x vector and transferred to DH5α strain of E. coli. Recombinant vector transformed and expressed into E. coli Rosetta strain. SDS-PAGE analysis indicated the production of a recombinant protein with an expected molecular weight of 75 KDa. Thereafter, the recombinant protein was purified by amylose resin and its antigenicity was accessed by immunoblotting. Positive reaction of the expressed protein with anti-trout serum indicated that the expressed constant region of trout μ chain possess antigenic epitopes and could be applied in future immunological studies.