Establishment And Application Of Detection Method Of Antimicrobial Activity Of Pseudo - Pectinase

In recent years, with the wide application of glucocorticoid and chemotherapy drugs, and the development of transplantation of solid organ and hematopoietic stem cell, the morbidity and mortality of invasive aspergillosis (IA) has significantly increased. Early diagnosis is key to the successful treatment of IA. However, the methods for diagnosis of IA has many disadvantages, such as the long detection period and low sensitivity, which make the method cannot meet the clinical need. Scholars in China and abroad are trying to find the biomarkers for the early diagnosis of IA. Research group screened a penal of immunodominant antigens, and found immune reactions between pectate lyase A (PlyA) and IA patients serum at the first time, which may be used as IA diagnostic marker.ObjectiveTo express Aspergillus fumigatus PlyA protein by prokaryotic expression system, establish an indirect ELISA method to detect IgG antibody against Aspergillus fumigatus PlyA in human serum, and evaluate the potential value for diagnosis of IA.MethodsThe gene sequence of Aspergillus fumigatus PlyA was optimized by bioinformatic analysis and selection of E. coli preferred codons. The synthesized DNA sequence was inserted into pET-28a(+) vector to construct the recombinant expression plasmid which was delivered our laboratory. The expression plasmid was transformed into E.coli BL21 (DE3), which expressed the PlyA recombinant protein by IPTG induction. The reactions of the recombinant protein with anti-His-tag antibody and the antibody in the serum from invasive aspergillosis patients were analyzed by Western blot. To establish an indirect ELISA method for detecting anti-PlyA antibody, and evaluate the value of the method in diagnosis of IA. Then, the diagnostic efficacy of the combined detection of anti-PlyA, anti-TR and anti-DPPV antibodies was analyzed.Results(1) The homology analysis showed that the Aspergillus fumigatus PlyA has no homology with the human proteins, and low homology with other common pathogen proteins. The optimized PlyA gene codon has changed in 18.84% of the nucleotide sequence, but the encoded amino acid sequence remains the same. The gene sequences was synthesized by a company, and the recombinant PlyA expression strains that can express the PlyA protein with His-tag by induction was successfully constructed.(2) Western blot result showed that the Aspergillus fumigatus PlyA can specifically react with anti-His-tag antibody and IA patients serum, but dosen’t react with Invasive Candidiasis patients serum and healthy control serum. The established the ELISA method shows good stability and specificity for detecting anti-PlyA antibody, which the intra-and inter-assay coefficient of variation (CV) of were 5.3% and 10.9%. The blocking rate of sera with recombinant PlyA was more than 90%.(3) The sensitivity and specificity of assay for IA patients were 51.5% and 94.3%, when A450=0.475 was definited as cutoff value. The positive rate of anti-PlyA antibody in non-neutropenic IA patients was significantly higher than in neutropenic IA patients. The positive rate of detection of combination with anti-PlyA, anti-TR and anti-DPPV antibodies can increase to 88.7%.ConclusionAn engineering strains, efficiently expressing Aspergillus fumigatus PlyA, was successfully prepared in this study. An ELISA method for detecting anti-PlyA antibody was developed and the potential value for the diagnosis of IA has been demonstrated.