Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

Abstract

Background: Mycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.

Methods: This study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni-NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.

Results: The resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.

Conclusion: The results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Keywords: Mycoplasma ovipneumoniae; Adhesin P113; Heat shock protein 70; I-ELISA.

Fig. 1

SDS‒PAGE detection of recombinant Hsp70‒P113 protein expression and purification. M: protein marker; 1: uninduced pET-30a/BL21(DE3); 2: uninduced pET-30a-rHsp70-P113/BL21(DE3); 3: induced pET-30a-rHsp70-P113/BL21(DE3); 4: supernatant of the induced pET-30a-rHsp70-P113/BL21(DE3); 5: precipitate of induced pET-30a-rHsp70-P113/BL21(DE3); 6: purified recombinant protein Hsp70-P113

Fig. 2

Western blot analysis of the reactogenicity of the recombinant protein Hsp70-P113. 1: Anti-His tag antibody as the primary antibody; 2: recombinant protein hyperimmune serum as the primary antibody (hyperimmune serum prepared by immunizing BALB/c mice with purified Hsp70-P113 recombinant protein); 3: Movi hyperimmune serum as the primary antibody (hyperimmune serum prepared by immunizing BALB/c mice with Movi whole bacterium antigen); 4. The serum of nonimmunized mice (blank control) was used as the primary antibody. M: protein marker. 1–4:HRP-labelled goat anti-mouse IgG was used as the secondary antibody

Fig. 3

Optimization of the ELISA. The concentration of the coating antigen (A), the dilution of the serum (B), and the dilution of the secondary antibody (C) were determined via the Checkerboard method

Fig. 4

Statistical frequency of negative sample results

Fig. 5

Specificity of the ELISA