[Expression of prn gene of Bordetella bronchiseptica and development of a recombinant protein-based indirect ELISA for antibodies detection].
OBJECTIVE
We developed an indirect ELISA method for detecting Bordetella bronchiseptica (Bb) pertactin antibodies based on the recombinant pertactin protein expressed in Escherichia coli (DE3) strain.


RESULTS
The prn gene encoding Bb pertactin was fused to the downstream of glutathione S-transferase (GST) of pGEX-KG expression vector, resulting in the fusion expression plasmid pGEX-prn. SDS-PAGE showed that the GST-PRN fusion protein was expressed in high level in BL21 carrying pGEX-prn. The strong reactivity of the GST-PRN fusion protein, specifically with antiserum against porcine Bordetellosis caused by Bb HH0809, was identified by Western blot. The recombinant protein fragment of rPRN was purified from the GST-PRN fusion protein digested by protease thrombin with the purity of 93.1%. The rPRN-based indirect ELISA was developed for detecting antibodies against PRN. The ELISA could detect positive samples in experimentally infected pigs fourteen days post inoculation and the degree of sensitivity was over 4 times higher than the latex agglutination test with the coating antigen of killed Bb. Thirty-two point seven percent of positive samples were detected in 1,229 clinical samples while no false positive results were found in detecting 7 antisera against porcine bacterial diseases. Sera samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method and the results indicated that pigs were infected by Bb during the nursery periods.


CONCLUSIONS
The assay showed excellent specificity, sensitivity and reduplication, and can be useful for epidemiological survey and clinical diagnosis of swine bordetellosis.
Zhao
Zhanqin
Z
State Key Laboratory of Agriculture Microbiology, College of Veterinary Medicine, Huazhong Agriculture University, Wuhan 430070, China. zhaozhanqin@126.com
Xue
Yun
Y
Wu
Bin
B
Tang
Xibiao
X
Chen
Huanchun
H
Li
Zengqiang
Z
Hu
Ruiming
R
Zhang
Jianmin
J
Duan
Longchuan
L
D000818:Animals; D000906:Antibodies / Q000032:analysis* / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015153:Blotting, Western; D016950:Bordetella bronchiseptica / Q000235:genetics*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D010566:Virulence Factors, Bordetella / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D000906:Antibodies; D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; C068929:pertactin