Molecular identification of Trichinella spiralis nudix hydrolase and its induced protective immunity against trichinellosis in BALB/c mice

Shao Rong Long, Zhong Quan Wang, Ruo Dan Liu, Li Na Liu, Ling Ge Li, Peng Jiang, Xi Zhang, Zi Fang Zhang, Hai Ning Shi, Jing Cui

BackgroundNudix hydrolases (Nd) is a widespread superfamily, which is found in all classes of organism, hydrolyse a wide range of organic pyrophosphates and has a `housecleaning¿ function. The previous study showed that Trichinella spiralis Nd (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with T7 phage-displayed TsNd polypeptides produced protective immunity. The aim of this study was to clone, express and identify the full-length TsNd and to investigate its immune protection against T. spiralis infection.Methodshe full-length cDNA sequence of TsNd gene encoding a 46 kDa protein from T. spiralis intestinal infective larvae (IIL) was cloned and identified. The antigenicity of recombinant TsNd protein (rTsNd) was analyzed by Western blot. Transcription and expression of TsNd at T. spiralis different developmental stages were observed by RT-PCR and Immunofluorescence test (IFT). The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd were determined by ELISA. The immune protection of rTsNd against T. spiralis infection in BALB/c mice was investigated.ResultsSequence and phylogenetic analysis revealed that TsNd had a nudix motif located at 226-244aa, which had high homology and the closest evolutionary status with T. pseudospiralis. An 89 kDa fusion protein, the cDNA clone (46 kDa) and MBP tag (43 kDa), was obtained after expression and purification. Western blot analysis showed that anti-rTsNd serum recognized the native TsNd protein with 46 kDa in crude antigens of muscle larvae (ML), IIL, adult worms (AW) and newborn larvae (NBL), and ES antigens of ML. Transcription and expression of TsNd gene was observed in all different developmental stages of T. spiralis (ML, IIL, AW and NBL), with high level expression in ILL. An immunolocalization analysis identified TsNd in the cuticle, stichocytes and reproductive organs of the parasite. Following immunization, anti-rTsNd IgG levels were increased, and the levels of IgG1 were more significantly higher than that of IgG2a. After a challenge infection with T. spiralis, mice immunized with the rTsNd displayed a 57.7% reduction in adult worms and a 56.9% reduction in muscle larval burden.ConclusionsTsNd induced a partial protective immunity in mice and could be considered as a novel candidate vaccine antigen against trichinellosis.