The DHFR amplification system has become very popular for use in developing recombinant CHO cell lines. SIgA showed neutralization activity against H5N1 virus on MDCK cells and the titer was decided to be 1?:?64. On preadministrating intranasally, the chimeric SIgA could prevent mice from lethal attack by using A/Vietnam/1194/04 H5N1 with a survival rate of 80%. So we concluded that the constructed recombinant chimeric SIgA has a neutralization capability targeting avian influenza virus H5N1 contamination in vitro and in vivo. 1. Introduction SC-26196 Endemic highly pathogenic avian influenza virus SC-26196 (AIV) H5N1 in poultry has been present since the first occurrence in 1997 in Hong Kong. AIV H5N1 circulates in waterfowl and domesticated avian species and has evolved into multiple phylogenetically distinct genotypes and clades [1C3], with geographically distinct groups in each country. H5N1 viruses occasionally infect humans, with high case-fatality rates. These viruses have repeatedly crossed the species barrier and caused highly lethal human infections. The wide distribution of highly pathogenic AIV H5N1 is usually a global threat to human health [4C7]. Most deaths have occurred in young, previously healthy, adults or children. According to the latest WHO report [8], there have been 633 laboratory-confirmed highly pathogenic H5N1 AI cases worldwide from 2003 to 2013, with a mortality of 59.6%. For active immunization, vaccination would be ideal; however, there are some problems with avian influenza (AI) vaccines at present. There is no current pandemic of AI in humans, and therefore it is difficult to accurately assess the protective effects of any vaccine. Vaccines also have a major drawback because it would take several weeks to produce protective antibodies. This often reduces preventative effects and obstructs their effectiveness as emergency protection, especially in some high-risk groups. In contrast, passive immune agents can make up for the deficiencies of vaccines and can generate protective effects immediately after administration. Research into passive immunity for AI prevention and treatment has been intensive in recent SC-26196 years. Animal experiments have shown that either polyclonal (serum, plasma) [9C11] or monoclonal [12C16] antibodies offer good protection against highly pathogenic AI. Meanwhile, many researchers have reported antibodies providing broad cross-protection against AIV H5N1 [12, 14, 17C19]. As a respiratory disease, AIV contamination occurs via respiratory or digestive tract mucosa. Secretory IgA (SIgA), first identified in the 1960s, is usually a type of IgA antibody found in breast milk, gastrointestinal fluids, respiratory secretions, and genitourinary tracts. SIgA consists of two monomeric IgA units, which are associated with the J chain acquired during the process of polymerization in plasma cells just before secretion, along with the secretory component (SC) [20, 21]. SIgA is considered the first-line defense in mucosal immunity and plays SC-26196 a critical role in preventing pathogen adhesion to host cells, thereby blocking dissemination and further contamination. Because of its dimeric structure, SIgA has a higher functional affinity [22]. In vitro, SIgA is usually more resistant to proteases than serum IgA [23C25]. Its half-life is usually three times longer than IgG on mucosal surfaces, and it can provide a specific protective effect for at least 4 months [22]. The presence of the SC also gives SIgA special protective immunity activity. First, the SC has nonspecific activity against pathogenic microorganisms [26]. Second, via carbohydrate residues, SIgA can adhere to epithelial surfaces, forming a protective layer and effectively preventing invasion by a virus [27, 28]. It would be of great significance to demonstrate the blocking effects of SIgA against AIV contamination in the respiratory or digestive tracts. Previous reports have shown that IgA can potentially be used for passive protection or therapeutic intervention on mucosal surfaces. IgA can act as a neutralizing antibody against pathogens and exotoxins, with better affinity than neutralizing antibodies of other classes [29]. Monoclonal IgA antibodies against respiratory syncytial virus were applied passively Mouse monoclonal antibody to RAD9A. This gene product is highly similar to Schizosaccharomyces pombe rad9,a cell cycle checkpointprotein required for cell cycle arrest and DNA damage repair.This protein possesses 3 to 5exonuclease activity,which may contribute to its role in sensing and repairing DNA damage.Itforms a checkpoint protein complex with RAD1 and HUS1.This complex is recruited bycheckpoint protein RAD17 to the sites of DNA damage,which is thought to be important fortriggering the checkpoint-signaling cascade.Alternatively spliced transcript variants encodingdifferent isoforms have been found for this gene.[provided by RefSeq,Aug 2011] to the nasopharyngeal mucosa and prevented SC-26196 subsequent contamination and pneumonia [30]. Passive oral delivery of IgA antibodies also guarded against bacterial infections in the intestine of mice [31]. IgA has lower proteolytic stability without the bound SC [23, 24], and therefore it may be efficient to use purified SIgA as a passive treatment agent. In this study, we constructed a mouse and human derived SIgA and explored its feasibility in preventing H5N1 virus contamination. Our results revealed that this recombinant SIgA could act as a preventative agent against H5N1 contamination. 2. Materials and Methods 2.1. Reagents, Cells, and Virus Restriction endonucleases and T4 Ligase for cloning were obtained from New England Biolabs (Beverly, MA, USA). Lipofectamine 2000 was purchased from Gibco BRL (Gaithersburg, MD, USA), with protein A-agarose, plasmid pcDNA4/His A, and zeocin from Invitrogen (Carlsbad, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM) and Fetal Bovine Serum (FBS) were obtained from Hyclone (Logan, UT, USA). A hybridoma cell line secreting an anti-H5N1 (HA).