The Preparation Of The Conservative Domains Of Ureaplasma Urealyticum MB Antigen And Its Monoclonal Antibodies For Diagnosis Purpose

Ureaplasma urealyticum (Uu) colonizes in lower genital tract of40-80%adults. It is not pathogenic; however, under some conditions, the organism causes several human diseases, e.g., non-gonococcal urethritis, prostatitis, infertility, premature delivery, fetus under weight, respiratory tract diseases. In the past50years, no conclusion was drawn on the relationships between Uu and those diseases, though numerous studies were carried out by using animal models, human experiments and epidemics methods. It is suggested that multi-factors, e.g., hosts’immunity, Uu species, antigenic variation, etc., were associated with or played important roles in the development of the diseases. Rapid diagnosis would be beneficial for the control of the diseases. To develop a colloidal gold immunochromatography test paper, the conservative domains of Ureaplasma urealyticum MB antigen and its monoclonal antibodies were prepared in this study.1. Cloning and expression of the gene encoding the conservative domains of MB (Ureaplasma urealyticum serum type Ⅲ), and analysis of the antigenicity of the rMBIn order to obtain the conservative domains of Uu MB antigen, we used EBI to analysis MB domains of14serotypes of Uu. The most homologous sequence of294bp gene fragment was selected to be cloned. Two pairs of primers were designed using oligo software to amplify the DNA fragment by polymerase chain reaction (PCR). The PCR product was inserted into pGEX-4T-1and pET-30a vector, respectively. Then we overexpressed GST-MB (37kD) and His-MB (11kD) in Escherichia coli BL21by adding IPTG as an inducer. The purified recombinant proteins were assayed by Bio-Rad protein purifier. The purification rates were 92.7%and86.7%, respectively. Furthermore, mice were immunized with His-MB and antiserum was prepared. Indirect ELIS A was performed to test the titer of the antibodies against MB in the immune sera using the recombinant GST-MB as antigen. The results indicate that the antiserum reacted with GST-MB from this study or that from a company. The antibody titer was between1:6400and1:12800. And the polyclonal antibodies reacted with Ureaplasma parvum serotypes3as well.2. The preparation of monoclonal antibodies against MB of Ureaplasma urealyticumTo prepare useful monoclonal antibodies of MB antigen, the recombinant His-MB obtained in part1was used to immunize BALB/c mice. Following4immunization, the titer of MB specific antibodies in the immune sera was detected to be1:12800by indirect ELIS A. The mice were sacrificed to harvest spleen cells. Then we fused myeloma cells (SP2/0) and spleen cells.6x96wells of cell cultures were performed limited dilution to get hybridoma cell lines which secreted MB specific monoclonal antibodies. We harvested3cell lines that secreted MB specific antibodies according to the data of indirect ELISA screening. Giemsa stain indicated that the hybridoma cells had95-105chromosomes after treated by colchicine. The cell lines were used to prepare ascites. Ascites were revealed to contain specific antibodies against MB by indirect ELISA. The titers of these antibodies were around1:320000; and the subtypes of them were IgG1. Western-blot showed that specific bindings appeared between the antibodies and the recombinant GST-MB. Furthermore, using the polyclonal antibodies and the monoclonal antibodies as primary antibody,50urine samples were tested by indirect ELISA. Accuracy of McAbs detection decreased significantly comparing with liquid cultivation (P<0.05) No difference was shown between polyclonal antibody detection and liquid cultivation (P>0.05).