Bioinformatic Prediction and Production of Four Recombinant Proteins from Different Developmental Stages of Trichinella spi-ralis and Testing of Their Diagnostic Sensitivity in Mice
Background: Trichinellosis is a serious food-borne parasitic zoonosis, thus finding high quality antigens is the key to serodiagnosis of trichinosis. This article reports the characterization and sensitivity of four recombinant proteins expressed by four genes (Wn10, Zh68, T668, and Wm5) from different developmental stages of Trichinella spiralis for the diagnosis of trichinellosis in mice.
Methods: This study was conducted in Jilin University and National Institute of Parasitic Diseases of Chinese Center for Disease Control and Prevention in 2017-2018. The structures and functions of the proteins encoded by four genes were predicted by bioinformatics analysis. The four genes were cloned and expressed, and the recombinant proteins were purified. Anti-Trichinella IgM and IgG antibodies in the sera of mice infected with T. spiralis from 1-45 d post-infection (dpi) were evaluated by ELISA.
Results: The optimal antigen epitopes of four proteins (P1, P2, P3, and P4) encoded by the four genes from T- and B-cells were predicted, and four purified recombinant proteins (r-P1, r-P2, r-P3, and r-P4) were successfully produced. For IgM, the antibody levels detected by the four recombinant antigens were approximately equal to the cut-off value. Anti-Trichinella IgG antibodies were first detected by r-P1 at 8 dpi, followed by r-P2, r-P3, and r-P4 at 10 dpi, 14 dpi, and 16 dpi, respectively, and the antibody levels remained high until 45 dpi.
Conclusion: The recombinant antigens r-P1, r-P2, r-P3, and r-P4 could be antigens that react with antibodies, they showed high sensitivity in the detection of anti-Trichinella IgG antibodies in mice. Among these proteins, r-P1 may be a candidate antigen for the detection of anti-Trichinella IgG antibodies in the early infection phase and exhibited the best sensitivity among the antigens.
2. Bruschi F. Trichinellosis in developing countries: is it neglected? J Infect Dev Ctries. 2012; 6(3):216-222.
3. Rostami A, Gamble HR, Dupouy-Camet J, et al. Meat sources of infection for out-breaks of human trichinellosis. Food Mi-crobiol. 2017; 64:65-71.
4. Murrell KD, Pozio E. Worldwide occur-rence and impact of human trichinellosis, 1986-2009. Emerg Infect Dis. 2011; 17(12):2194-2202.
5. Murrell KD. The dynamics of Trichinella spiralis epidemiology: out to pasture? Vet Parasitol. 2016; 231:92-96.
6. Gottstein B, Pozio E, Nöckler K. Epide-miology, diagnosis, treatment, and control of trichinellosis. Clin Microbiol Rev. 2009; 22(1):127-145.
7. Zolfaghari Emameh R, Kuuslahti M, Näreaho A, et al. Innovative molecular di-agnosis of Trichinella species based on β-carbonic anhydrase genomic sequence. Microb Biotechnol. 2016; 9(2):172-179.
8. Li X, Liu W, Wang J, et al. Rapid detection of Trichinella spiralis larvae in muscles by loop-mediated isothermal amplification. Int J Parasitol. 2012; 42(13-14):1119-1126.
9. Gómez-Morales MA, Ludovisi A, Amati M, et al. A distinctive western blot pattern to recognize Trichinella infections in hu-mans and pigs. Int J Parasitol. 2012; 42(11):1017-1023.
10. Yang Y, Cai YN, Tong MW, et al. Serologi-cal tools for detection of Trichinella infec-tion in animals and humans. One Health. 2016; 2:25-30.
11. Shimoni Z, Froom P. Uncertainties in di-agnosis, treatment and prevention of trichinellosis. Expert Rev Anti Infect Ther. 2015; 13(10):1279-1288.
12. Yera H, Andiva S, Perret C, et al. Devel-opment and evaluation of a western blot kit for diagnosis of human trichinellosis. Clin Diagn Lab Immunol. 2003; 10(5):793-796.
13. Gómez-Morales MA, Ludovisi A, Amati M, et al. Validation of an enzyme-linked immunosorbent assay for diagnosis of human trichinellosis. Clin Vaccine Immu-nol. 2008; 15(11):1723-1729.
14. Mitreva M, Jasmer DP, Zarlenga DS, et al. The draft genome of the parasitic nema-tode Trichinella spiralis. Nat Genet. 2011; 43(3):228-235.
15. Liu MY, Wang XL, Fu BQ, et al. Identifi-cation of stage-specifically expressed genes of Trichinella spiralis by suppression subtractive hybridization. Parasitology. 2007; 134:1443-55.
16. Wang ZQ, Shi YL, Liu RD, et al. New in-sights on serodiagnosis of trichinellosis during window period: early diagnostic an-tigens from Trichinella spiralis intestinal worms. Infect Dis Poverty. 2017; 6(1): 41.
17. Zhai CC, Sun ZJ, Liu MY, et al. Kinetics evaluation of IgM and IgG levels in the mice infected with Trichinella spiralis exper-imentally using ES antigens from different developmental stages of the parasite. Iran J Parasitol. 2019; 14(2):223-230.
18. Wu XP, Fu BQ, Wang XL, et al. Identifica-tion of antigenic genes in Trichinella spiralis by immunoscreening of cDNA libraries. Vet Parasitol. 2009; 159(3–4):272-275.
19. Tang B, Liu M, Wang L, et al. Characteriza-tion of a high-frequency gene encoding a strongly antigenic cystatin-like protein from Trichinella spiralis at its early invasion stage. Parasit Vectors. 2015; 8:78.
20. Yang Y, Wen YJ, Cai YN, et al. Serine pro-teases of parasitic helminths. Korean J Parasitol. 2015; 53(1):1-11.
21. Mayer-Scholl A, Pozio E, Gayda J, et al. Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples. J Vis Exp. 2017; (121): 55354.
22. Wang L, Wang X, Bi K, et al. Oral vaccina-tion with attenuated Salmonella typhimurium-delivered TsPmy DNA vaccine elicits pro-tective immunity against Trichinella spiralis in BALB/c mice. PLoS Negl Trop Dis. 2016; 10(9): e0004952.
23. Office International des Epizooties. Trichinellosis, chapter 2.2.9. In Manual of standards for diagnostic tests and vaccines, 5th ed. 2004; Office International des Epi-zooties, Paris, France.
24. Bien J, Cabaj W, Moskwa B. Proteomic analysis of potential immunoreactive pro-teins from muscle larvae and adult worms of Trichinella spiralis in experimentally in-fected pigs. Folia Parasitol (Praha). 2015; 62:2015.022.
25. Liao C, Liu M, Bai X, et al. Characteriza-tion of a plancitoxin-1-like DNase II gene in Trichinella spiralis. PLoS Negl Trop Dis. 2014; 8(8): e3097.
26. Wang L, Cui J, Hu DD, et al. Identification of early diagnostic antigens from major excretory-secretory proteins of Trichinella spiralis muscle larvae using immunoprote-omics. Parasit Vectors. 2014; 7:40.
27. Sun GG, Wang ZQ, Liu CY, et al. Early serodiagnosis of trichinellosis by ELISA using excretory-secretory antigens of Trichinella spiralis adult worms. Parasit Vec-tors. 2015; 8:484.
28. Sun GG, Liu RD, Wang ZQ, et al. New diagnostic antigens for early trichinellosis: the excretory-secretory antigens of Trichi-nella spiralis intestinal infective larvae. Para-sitol Res. 2015; 114(12):4637-4644.
29. Hu CX, Jiang P, Yue X, et al. Molecular characterization of a Trichinella spiralis elas-tase-1 and its potential as a diagnostic anti-gen for trichinellosis. Parasit Vectors. 2020; 13(1):97.
30. Xu J, Bai X, Wang LB, et al. Immune re-sponses in mice vaccinated with a DNA vaccine expressing serine protease-like protein from the new-born larval stage of Trichinella spiralis. Parasitology. 2017; 144(6):712-719.
31. Zocevic A, Lacour SA, Mace P, et al. Pri-mary characterization and assessment of a T. spiralis antigen for the detection of Trichinella infection in pigs. Vet Parasitol. 2014; 205(3-4):558-567.
|Issue||Vol 16 No 1 (2021)|
|Trichinella spiralis Genes Recombinant protein Bioinformatics analysis Diagnostic characteris-tics ELISA|
|Rights and permissions|
|This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.|