Prokaryotic Expression Of P17 Gene Of Bartonella Henselae And Establishment Of An Indirect ELISA For Detection Of Expression Product

Purpose: For check out the pathogen of Bartonella henselae by indirect ELISA method in clinically, established the optimum condition of indirect ELISA method using the recombination protein which was inducted expression of Bartonella henselae P17 gene.Methods: According to the gene sequences in Genebank, amplified the P17 by PCR, which is the unique gene of B. henselae (the size as 525bp), and cloned the gene to the vector pET-28a. Then transformed the recombinant expression plasmid into the E. coli DH-5αand induced the P17 protein expression by IPTG with a finial concentration 1mmol/L, and using the purified interest protein with the assay of Ni-NTA column as antigen established the Bartonella henselae indirect Elisa method in human clinically.Results: Successfully obtained, the Molecular Weight was 22.6KD and the concentration was 3.44mg/ml, the recombinant protein of P17 gene. Moreover, established the optimum condition of indirect ELISA method: dilute concentration of interest protein of P17 gene as 1.25μg/mL coated 37℃for 2hour, closed 1hour at 37℃using 5% separated milk, testing serum dilution at l: 400 incubated 1 hour at 37℃, and using the HRP-Goat anti-human IgG dilution at l: 5000 to incubate 1 hour at 37℃, then the substrate reacted 25 minutes at 37℃and protection from light. Finally gain the yin-yang critical value of ELISA was 0.1238. Using the method of the established ELISA measured clinical serum samples of 91 from Shanghai and 393 from Liao Ning province. And the results showed that the ratio of the positive was 24.17% and 19.59% respectively.Conclusion:Established Bartonella henselae indirect ELISA method successfully in human clinically, the method was sensitive and specific. Besides, the method ,using prokaryotic expression obtained the recombination and purified protein as antigen, has the characteristic such as low cost, convenient operation, creditable consequence and so on, which could supply reliable experiment accordance and rationale for parturient practice and clinical diagnose using the establish ELISA method in the study.