Bioinformatic Prediction and Production of Four Recombinant Proteins from Different Developmental Stages of Trichinella spiralis and Testing of Their Diagnostic Sensitivity in Mice

Abstract

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.

Keywords: Bioinformatics analysis; Diagnostic characteristics; Genes; Recombinant protein; Trichinella spiralis.

Fig. 1:

Hydrophobicity and hydrophilicity of four proteins of T. spiralis analyzed by ExPASy-ProtScale (K-D methods). a: P1; b: P2; c: P3; d: P4

Fig. 2:

Transmembrane region of four proteins of T. spiralis analyzed by TMHMM. a: P1; b: P2; c: P3; d: P4

Fig. 3:

Structure domain of P2, P3 and P4 of T. spiralis analyzed by SMART. a: P2; b: P3; c: P4.

Fig. 4:

Secondary structure of four proteins of T. spiralis predicted by ExPASy-SOMPA. a: P1; b: P2; c: P3; d: P4

Fig. 5:

Tertiary structure of four proteins of T. spiralis predicted by the SWISS-MODEL. a: P1; b: P2; c: P3; d: P4

Fig. 6:

Total RNA and gene amplification M: DL2000 Maker; 1: Total RNA of Trichinella spiralis; 2: Wn10 gene amplification; 3: Zh68 gene amplification; 4: T668 gene amplification; 5: Wm5 gene amplification

Fig. 7:

SDS-PAGE analysis of r-P1, r-P2, r-P3 and r-P4 of T. spiralis M: Marker; 1: pET-28a without IPTG induction; 2: pET-28a induction with IPTG for 4h; 3: pET-Wn10 without IPTG induction; 4: pET-Wn10 induction with IPTG for 4h; 5: r-P1 after purification; 6: pET-Zh68 without IPTG induction; 7: pET-Zh68 induction with IPTG for 4h; 8: r-P2 after purification; 9: pET-T668 without IPTG induction; 10: pET-T668 with IPTG induction for 4h; 11: r-P3 after purification; 12: pET-Wm5 without IPTG induction; 13: pET-Wm5 with IPTG induction for 4h; 14: r-P4 after purification

Fig. 8:

The levels of anti-Trichinella IgM antibodies in the sera of mice infected with four doses of muscle larvae a: r-P1; b: r-P2; c: r-P3; d: r-P4

Fig. 9:

The levels of anti-Trichinella IgG antibodies in the sera of mice infected with four doses of muscle larvae a: r-P1; b: r-P2; c: r-P3; d: r-P4