## 1 Introduction  
Antimicrobial peptides are essential effector molecules that provide the host immune response against pathogen challenge. To date, antimicrobial peptides have been shown to have activities against infection from a wide range of microorganisms including bacteria, fungi, viruses and other pathogens [1; 2; 3; 4; 5; 6]. [PERSON] discovered the first antimicrobial peptide from _Hyalophon cercopia_ in 1972, namely ecropins [7]. Nearly 1500 antimicrobial peptides were gradually identified from life entities including bacteria, fungi, plants, insects, fish, amphibians, birds and mammals [8; 9; 10; 11; 12; 13]. Fish, as an indispensable organism in water, also possess numerous AMPs (antimicrobial peptides). Since then, over 100 fish AMPs have been isolated, artificially synthesized or expressed by genetic engineering [14], including catheclicidin, chrysophsin, hepcidin, hipposin, misgurin, moroncoidin, liver expressed antimicrobial peptide 2 (LEAP-2), oncorhynchus, parasin, pardaxin, puscidin, pleurcoidin, NK-lysin [14; 15; 16; 17; 18; 19; 20]. According to different structural features, fish antimicrobial peptides can be broadly classified into four classes. These four classes are \"traditional\" classes that have had comprehensive studies through recent years. In addition to the previously known four classes, the fifth class of antimicrobial polypeptides has been identified [21] and it includes NK-lysin [22], HDL [23], misgurin [24] and myxidin [25]. NK-lysin was first identified from porcine natural killer cells and cytotoxic T lymphocytes based on its antibacterial activity [26]. Compared with the first four classical antimicrobial peptides, NK-lysin is much larger with 74-78 amino acid residues than the former AMPs [27]. It possesses six half-cystine residues in three intrachain disulphide bonds [28]. A study shows that porcine NK-lysin has strong antibacterial activity against _Escherichia coli_ and _Bacillus megaterium_, and it also exerts activity towards an NK-sensitive tumor cell line of murine animals [26]. However, the porcine NK-lysin has no lytic activity against erythrocytes [26; 28]. Artificially synthesized Japanese flounder NK-lysin has been found to have activity against various Gram-negative bacteria [29]. Until now, various animals' NK-lysin gene sequences have been identified including mammalian species such as cow [30], pig [26] and horse [31]; birds such as chicken [32]; and aquatic animals such as channel catfish [33], zebrafish [34] and _Cynoglossus semilaevis_[35]. Three distinct NK-lysin transcripts have been identified in channel catfish, respectively NK-lysin 1, NK-lysin 2, NK-lysin 3 [36]. They are highly expressed in gill, head kidney, spleen and trunk kidney, but are incapable of being expressed in all tissues, and all three genes have different expression profiles [36; 37]. Previously, it was found that the expressions of NK-lysin 1, NK-lysin 2 and NK-lysin 3 were all up-regulated from the second or third day to the seventh day or even longer after being infected by _Edwardsiella ictaluri_[36; 38]. Therefore, NK-lysin is an important component of the immune response of channel catfish and possesses the potential of against pathogen challenge. With the development of biotechnology, the genetic recombinant technique has become a more effective way to produce drug proteins and polypeptides than traditional isolation or artificial synthesis methods. Many AMPs have been expressed in heterologous hosts, especially with the _Escherichia coli_ expression system [39]. The _E. coli_ system is widely accepted for the use of heterologous protein production [40]. It is a high-yield and reliable system because the _E. coli_ can survive in a wide range of environmental conditions and the system can be easily controlled. However, AMPs have high toxicity towards host strains so that different molecular chaperones are used in order to reduce toxicity and inclusion bodies to improve fusion protein expression [41].