Expression Of MB Antigen Conserved Region Of Ureaplasma Urealyticum In E. Coli And Preparation Of Antibodies Of MB Antigen And Apply To The MB-antibody Probe Uu In The Clinic Urines Samples

Objective To establish foundation for preparation diagnostic kit of the Uu assay, the recombinant plasmid of encoding partial conserved segment of MB antigen of Ureaplsama urealyticum serotype 1 (Uu1) was constructed. And MB antigen gene segment was expressed in E. coli. The expression production was purified with affinity chromatography, and the high specific antibody to the MB antigen was prepared through immunization of NewZealand white rabbit. The specificity and sensitivity of the MB-antibodies that probed the Uu in clinic samples were tested.Methods The immunodominant epitope of the encoding partial conservedsegment of MB antigen of Uul was chosen by computer analysis, and the MB segment was amplified from Uul complete genome by polymerase chain reactions (PCR) and cloned into the expression vector pQE30. The recombinant plasmid pQE30/partMBA was transformed into E. coli M15. The induced fusion protein 6His-partMBA was analyzed by SDS-PAGE and Western-Blot, and was purified with Ni Chelating Sepharose F.F. affinity chromatography. The concentration of the purified protein was determined by the BCA protein assay kit, and the fusion protein was used to immunize New Zealand white rabbit. The anti-serum was evaluated by indirect-ELISA and the immunogenicity of the fusion protein was analyzed. The polyclonal antiserum was used to detect Uu in the clinical urines samples by indirect-ELISA.Results Analyse the antigenicity, hydrophobicity and transmembrane domain ofthe MB antigen using software, and choice the part of conserved segment from the 208bp to 540bp of the MB-antigen gene. The length of target gene is 333bp and...