Production and characterization of monoclonal antibodies against YopM effector protein of Yersinia pestis.
The YopM is an essential virulence effector produced by the bubonic plague bacterium. Yersinia pestis specific PCR gene was developed using 780 bp fragment of yopM gene. The PCR product was further cloned (in pUC57) an subcloned (pQE32 expression vector) and transformed in SG13009 E. coli host cells. The IPTG-induced recombinant protein was expressed at approximately 32 kDa region by SDS-PAGE. The recombinant protein was with 80% purity and 3mg/mL of concentration. Polyclonal and monoclonal antibodies (MAb) were generated. A total number of nine specific monoclonal antibodies obtained reacted at 43 kDa native protein of Y. pestis. Both the PCR-based assay and immunoassays were evaluated on Indian Y. pestis strains. Isolates recovered from outbreak region were positive, whereas isolates recovered from the surveillance region were negative (except one) by yopM gene PCR- and MAb-based dot-ELISA. The PCR- and ELISA-based systems developed in the present study might be utilized for detection or strain typing of Y. pestis alone or in conjunction with virulence markers such as F1 (fraction 1) and Pla (plasminogen activator).
Khushiramani
Rekha
R
Division of Microbiology, Defence Research and Development Establishment, Gwalior, India.
Tuteja
Urmil
U
Shukla
Jyoti
J
Batra
Harsh Vardhan
HV
D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000302:isolation & purification*; D000942:Antigens, Bacterial / Q000276:immunology; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas; D007194:India; D051379:Mice; D008807:Mice, Inbred BALB C; D010930:Plague / Q000175:diagnosis / Q000276:immunology; D011817:Rabbits; D037521:Virulence Factors; D015010:Yersinia pestis / Q000276:immunology*
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D037521:Virulence Factors; C060772:yopM protein, Yersinia