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 Mycoplasma ovipneumoniae (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. Afterward, 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 through SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the aforementioned protein as the coating antigen. The specificity, sensitivity, and reproducibility of the method were assessed after optimizing each reaction parameter. 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 recombinant rHsp70-P113 protein was encapsulated at a concentration of 5 µg/mL, the serum to be tested was diluted at a ratio of 1:50, and the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The cross-reactivity assays demonstrated that the i-ELISA was not cross-reactive with other goat-positive sera against orf virus (ORFV), Mycoplasma mycodies subsp. capri (Mmc), or Enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of samples of up to 1:640. The results of intrabatch and interbatch replication tests showed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples using the i-ELISA and indirect haemagglutination techniques yielded significant findings. Of these samples, 43 displayed positive results, while 65 showed 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 the Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.