Zastosowanie metody inżynierii genetycznej do uzyskania białka P1 Mycoplasma pneumoniae oraz ocena jego przydatności w serodiagnostyce mykoplazmozy u ludzi
[Use of recombinant P1 protein of Mycoplasma pneumoniae for the serodiagnosis of mycoplasmosis]

Streszczenie

Metodami inżynierii genetycznej uzyskano preparat białka P1 M. pneumoniae oraz oceniono jego przydatność w serodiagnostyce mykoplazmozy u ludzi. Wyniki oznaczeń metodą ELISA poziomu mykoplazmowych przeciwciał przy użyciu tego białka porównano z wynikami oznaczeń uzyskanymi w odczynie wiązania dopełniacza oraz w komercyjnym teście western-immunoblotting firmy Virotech. Przeprowadzone badania wykazały, że zastosowana metoda pozwoliła uzyskać wysoce oczyszczone białko, które może być z powodzeniem wykorzystane jako swoisty antygen w odczynach serologicznych prowadzo­nych w kierunku zakażeń wywoływanych przez M. pneumoniae.

Abstract

Introduction. Mycoplasma pneumoniae is a common etiologic agent of community-acquired respiratory infection. In serological diagnosis of M. pneumoniae infections tests have been described based on the purified P1 protein, which is the most important virulence factor of this pathogen, as antigen. The aim of his study was to express and purify a recombinant protein P1 M. pneumoniae and next evaluate this protein as high specific antigen in sero­diagnosis of mycoplasmosis. Methods. Protein P1 M. pneumoniae was expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Based on published literature, we decided to express C-terminal region [P1-C1] encompassing amino acid residues 1160 to 1521. Purification was accomplished by immobilized metal (Ni2+) affinity column chromatography (His-trap). Serum samples collected from 221 patients with mycoplasmosis, positive in complement fixation test (CFT), 87 patients with other then mycoplasmosis bacterial infections and 80 blood donors were screened for anti-P1 recombinant protein IgA, IgG and IgM antibodies by using the home-made ELISA. Results. SDS-PAGE and Coomassie brilliant blue staining confirmed a high purity of the recombinant P1 protein preparation with an expected molecular mass of 39,7 kDa. The specificity of the recombinant protein was confirmed by western blot analysis using serum samples from rabbits immunized by M. pneumoniae. The results of ELISA revealed that more then 70.0% of patients with mycoplasmosis confirmed by CFT, had antibodies to re­combinant P1 protein in diagnostically significant level (x+2SD). The antibodies were found only sporadically in sera obtained from patients with other then mycoplasmosis bacterial infections and clinically healthy persons. A comparison of results obtained in home-made ELISA with results of commercial western blot (Virotech) showed similar, ranged from 84.2% to 97.4%, compatible of results. The IgM antibodies to recombinant P1 protein were found in 87.2% sera obtained in acute phase of disease, in 80.0% sera obtained 2-4 weeks after onset of clinical symptoms and only in 43.8% sera obtained in chronic mycoplasmosis. Conclusions. The present study confirmed the earlier observations of the high usefulness of recombinant P1 protein for reliable serologic diagnosis of M. pneumoniae infection.