[Establishment and application of enzyme linked immunosorbent assay based on the outer membrane pIA-pIB fusion gene of Neisseria gonorrhoeae].
OBJECTIVE
To clone pIA and pIB genes of Neisseria gonorrhoeae,and to construct pIA-pIB fusion gene and its prokaryotic expression system, and to establish enzyme linked immunosorbent assay (ELISA) based on rPIA-PIB for detecting serum and pus samples from gonorrhea patients and to evaluate the sensitivity and specificity of the ELISA.


METHODS
pIA-pIB fusion gene was constructed by polymerase chain reaction (PCR) using linking primers and a prokaryotic expression system of the fusion gene was constructed by using routine molecular biological methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) plus BioRad Gel Image Analyzer was used to measure the expression of the target recombinant protein rPIA-PIB. Ni-NTA affinity chromatography was performed to extract and purify rPIA-PIB. An ELISA by using rPIA-PIB as the coated antigen for detecting the specific IgG against rPIA and/or rPIB in gonorrhea patients' sera as well as another ELISA by using rPIA-PIB antiserum as the first antibody for detecting the rPIA and/or rPIB in gonorrhea patients' pus samples were established. In these experiments, ELISAs associated with rPIA, rPIB and their antisera were applied as the controls.


RESULTS
100% similarities of the nucleotide and putative amino acid sequences of the pIA-pIB fusion gene were confirmed when compared with the original sequences. The output of rPIA-PIB was 29.8% of the total bacterial proteins. The purified rPIA-PIB only showed a single target protein segment in gel after SDS-PAGE. Using a positive rate (98.3%) of rPIA-PIB-IgG-ELISA to detect 119 cases of gonorrhea patients' serum samples was remarkably higher than that of rPIA-IgG-ELISA (30.3%) or rPIB-IgG-ELISA (66.4%) (P<0.01). The positive rate (91.6%) of rPIA-PIB-ELISA to detect 119 cases of gonorrhea patients' pus samples was also significantly higher than that of rPIA-IgG-ELISA (27.7%) or rPIB-IgG- ELISA (62.2%) (P<0.01).


CONCLUSIONS
In this study we successfully constructed pIA-pIB fusion gene of N. gonorrhoeae and its prokaryotic expression system while rPIA-PIB showed obvious superiority used as the antigen in gonorrhea associated detection kits compared to both the rPIA and rPIB.
Sun
Ai-Hua
AH
Faculty of Basic Medicine, Zhejiang Medical College, Hangzhou, China.
Fan
Xing-Li
XL
Yan
Jie
J
D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009344:Neisseria gonorrhoeae / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins