关键词: 旋毛虫, 新生幼虫, 免疫蛋白组学, LC-MS/MS, GO注释

Abstract: Objective To identify the soluble antigens of Trichinella spiralis newborn larvae recognizable by the serum of mice infected with T. spiralis for screening candidate antigens of anti-newborn larvae vaccines. Methods The adult worms were collected from the intestine of T. spiralis-infected BALB/c mice and cultured to collect the newborn larvae, of which the soluble antigens were extracted for screening out the antigen band recognized by sera from T. spiralis-infected mice using Western blotting. The recognized antigen on the blotting band was identified by liquid chromatograph tandem mass spectrometer (LC-MS/MS), and aligned with data of T. spiralis in the Uniprot database. The physicochemical properties of identified proteins were analyzed using the bioinformatics online website. The InterProscan software was used to perform proteins sequences searches against InterPro member databases to identify signatures, and the matched terms were further subjected to gene ontology (GO) categorizing using WEGO online software. The worms at different developmental stages including muscle larvae, infectious intestinal larvae (6 h post-infection), adult worms (2 d post-infection) and newborn larvae were collected to extract total RNA,which was then reversely transcribed into cDNA. The relactive transcription levels of the C-type lectin (CTL), calreticulin (CRT), zinc finger protein (ZFP) and pyruvate kinase (PK) at four developmental stages were analyzed by qPCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as the internal reference and compared with their transcription level at muscle larval stage using one-way ANOVA. Results Western blotting showed that among the 11 protein bands of the soluble antigens of newborn larvae, 4 bands [the relative molecular mass (M_r) were 99 700, 79 600, 68 900 and 46 000] were recognized by T. spiralis-infected mice sera. A total of 353 T. spiralis proteins were identified by LC-MS/MS, of which 166 proteins (47.0%) had M_r 40 000-70 000, 182 proteins (51.8%) had isoelectric point of 5-6, 31 proteins had signal peptides and 58 proteins had transmembrane domains. Out of the 353 proteins, 285 proteins had the GO annotations, of which 177 proteins (62.1%) had catalytic activity, 192 proteins (67.4%) had binding activity, 158 proteins (55.4%) were involved in the metabolic process and 149 proteins (52.3%) were involved in the cellular process. The qPCR results showed that compared to those at the muscle larvae stage, the relative transcript level of CTL at infectious intestinal larvae, adult worm and newborn larvae stages were 140.99%, 90.99% and 65.71%, respectively (F = 1 875.105, P ˂ 0.01); the CRT were 79.33%, 41.59% and 58.58%, respectively (F = 2 192.665, P ˂ 0.01); the ZFP were 64.93%, 105.36% and 126.74%, respectively (F = 475.836, P ˂ 0.01); the PK were 73.93%, 98.09% and 43.19%, respectively (F = 1 373.743, P ˂ 0.01). Conclusion A total of 353 soluble proteins of T. spiralis newborn larvae recognized by T. spiralis-infected mice sera were identified, and these proteins may involve in the processes of growth and development, immune evasion, and invasion of the host etc, and might be the candidate target molecules for anti-newborn larvae vaccines.

Key words: Trichinella spiralis, Newborn larvae, Immunoproteomics, LC-MS/MS, GO categories