Screening And Identification Of The Specific Protein Antigen Of Klebsiella Pneumonia And Pasteurella Pneumotropica

Klebsiella pneumonia and Pasteurella pneumotropica are the common pathogenic bacteria of laboratory animals especially for mice and rats. The infection of these becteria of laboratory animals will seriously affect the results of experiments; therefore they are obliged to be excluded for specific pathogen free (SPF) animals according to the national standard. Currently they were detected by separating and culturing, and the method should be improved due to low sensitivity. Serologic assay based on specific antigen has the advantage of high sensitivity, specificity, low cost, timesaving and the capability of diagnosing animals in convalescence after infection, and it is a preferred way for infection detection of laboratory animals.In our research of specific antigen of K. pneumonia, polyclonal antibodies of 10 common pathogenic bacteria that mice often-infected were made by immunizing mice with bacteria bodies inactivated by formalin. The two-dimension electrophoresis (2DE) was performed to separate total protein of K. pneumonia. Strong immunogens were selected by western blotting with its own polyclonal antibodies, while immune cross-reactions were excluded by reaction to other polyclonal antibodies referred before. Specific antigens were selected and identified by mass spectrum (MS). We have discovered a protein with high sensitivity and specificity in western blotting. It is identified as Acid phosphatase (Gi: 238894261) in MS.The coding sequence of this antigen protein was obtained in NCBI genome database. Primers were desigened to amplify the gene of the antigen. The gene was ligated to an expression vector. The protein was expressed in E.coli. and then purified. The sensitivity and specificity was certified by Enzyme-Linked Immunosorbent Assays (ELISA). The sensitivity was evaluated by signal-to-noise value, that is, mean absorbance of positive serum vs that of cross reactions. We have got a signal-to-noise ratio of 3.25 of this antigen. The result has certified that Acid phosphatase was the specific protein antigen for K. pneumonia.However, the genome information of P. pneumotropica is still unknown. An alternative method was tried by screening the protein in the closest species, Pasteurella multicida. Sensitive antigen was selected from P. multicida total protein by reacting to P. pneumotropica pAbs and cross reaction was excluded by hybridizing to other pAbs. finally, we got an antigen PM1693 (Gi:15603558), a hypothetical protein, with high sensitivity and specificity. The signal-to-noise value is 2.36. Furthermore, mice are not susceptible to P. multicida. This protein was expected to be a candidate protein antigen for P. pneumotropica diagnosis.