Application of gene recombination and chromatography of bio-affinity for production of the purified Yersinia enterocolitica protein antigens use of the recombinant YopD protein in serodiagnosis of enteric illness and reactive arthritis
Abstract number: 902_p1634
Yersinia enterocolitica causes a variety of infections in human, including enterocolitis, acute mesenteric lymphadenitis, erythema nodosum and reactive arthritis. Both whole Yersinia bacteria and purified lipopolysaccharide (LPS) have been used as antigens for detecting antibodies in sera of patients. However cross-reactions in the ELISA between Y. enterocolitica and other pathogens have been described. Therefore high specific antigens, free of lipopolysaccharide, based on Yersinia outer membrane proteins (YOPs), or adhesins: Ail, YadA, Invasin or Myf are needed. The aim of this study was to evaluate the usefulness of gene recombination technique using the pET-30 Ek/LIC expression vector for production a 36-kDa YOP called YopD and evaluate of this purified protein as antigen in serodiagnosis of yersiniosis.
Protein YopD of Y. enterocolitica was expressing in Escherichia coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with Bug Buster Protein/Extraction Reagent was accomplished by immobilised metal (Ni2+) affinity column chromatography (His-trap). The IgM, IgG and IgA class antibodies to YopD were measured in 100 serum samples collected from patients suspected for yersiniosis and 100 blood donors. The obtained results were compared with the results of ELISA with LPS and YOPs isolated from the culture of Y. enterocolitica supernatant under calcium deficient conditions, as antigens.
In the case of patients suspected in clinical investigation for yersiniosis most frequently the positive results were obtained in IgG class of antibodies (41.0%). IgM and IgA antibodies were detected in 33.0 and 10.0% serum samples, respectively. In sera obtained from blood donor's antibodies, in all immunoglobulin classes, to YopD antigen were detectable significantly rarely (P < 0.001). A very high (94.0100.0%) specificity and good sensitivity (67.080.0%) was displayed by the ELISA with YopD in relation to ELISA with LPS and YOPs antigens. IgA and IgG more frequently were found in sera of adult persons with arthritis and immunoglobulin M in the sera of children with enteric illness.
The recombinant YopD protein purified by chromatography of bio-affinity may be used in serodiagnosis of yersiniosis as a high specific antigen free of Yersinia lipopolysaccharides."
|Session name:||XXIst ISTH Congress|
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