ABSTRACT
The purified elementary bodies of C. pneumoniae TW-183 were used for immunization of male BALB/c mice, the spleen cells of these mice were fused with SP2/0 cells and the hybrid cells were cloned by limiting dilution. One clone that secreted the C. pneumoniae monoclonal antibody (Cpn-McAb) stably was obtained finally. The Cpn-McAb belonged to IgG2b class and anti-Cpn-MOMP; the outcome of micro-immunofluorescence showed its weak cross reaction with the C. psittaci elementary body but it has no cross reaction with C. trachoma elementary body. It has the same speciality of the imported Cpn-McAb. For the evaluation of Cpn-McAb, the peripheral blood mononuclear cell specimens of 454 patients were detected by self-made Cpn-McAb and imported Cpn-McAb at the same time. The positive rates of Cpn-antigen were 53.3% for self-made Cpn-McAb and 52.6% for imported Cpn-McAb,showing high concordance between them (Kappa=0.714). The results showed that self-made Cpn-McAb has almost the same high specificity and sensitivity as imported Cpn-McAb, so the self-made Cpn-McAb may replace imported Cpn-McAb to detect Cpn specific antigen and be helpful to diagnosing and treating the clinical diseases associated with Cpn infection.
Subject(s)
Animals , Male , Mice , Antibodies, Bacterial , Allergy and Immunology , Antibodies, Monoclonal , Genetics , Antibody Specificity , Chlamydia Infections , Diagnosis , Microbiology , Chlamydophila pneumoniae , Allergy and Immunology , Hybridomas , Bodily Secretions , Mice, Inbred BALB CABSTRACT
Objective To obtain staphylococcal enterotoxin B (SEB) mutant with normal antigenicity but low toxicity. Methods Using PCR technique, normal SEB (SEB-N) gene which was amplified from S. aureus S6B. SEB mutant gene (SEB-M) was prepared from the same strain, but one nucleotide in SEB gene was changed from asparagine (N23) to serine (S23). SEB-N and SEB-M were cloned into procaryotic expression vector pTrc99A then and transferred into E. coli JM109. SEB-N and SEB-M which were cloned into plasmid were sequenced directly by dideoxynucleotide method. The crude expressed proteins were identified by double agar immunodiffusion. The level of IL-2 in supernatants of mouse splenocytes stimulated by crude expressed proteins was determined by ELISA. Results SEB-N and SEB-M were obtained through PCR. The sequence of SEB-N was changed with non site-directed mutagenesis, threonine at the residue 150 of SEB-N was replaced with alanine (ACT→GCT, T150A). As being expected, at the residue 23 of SEB-M, serine substituted for asparagine (AAT→AGT, N23S) with site-directed mutagenesis. Double agar immunodiffusion showed obvious precipitin line with anti-SEB by both crude SEB-N and SEB-M mutant proteins could produce, but not by non-recombinant strain. ELISA demonstrated that the level of IL-2 in supernatant of mouse splenocytes stimulated by natural SEB protein (containing equal amount of JM109P crude protein) was 40 times as much as that stimulated by SEB-M and 12.5 times as much as that stimulated by SEB-N. Conclusions We obtained two recombinant strains which produced T150A and N23S mutant SEB protein. The mutant proteins showed binding ability to anti-SEB as the normal protein. However, their biological activity as superantigen decreased sharply. We consider that it is promising for further study of molecular adjuvant or superantigen vaccine.
ABSTRACT
The anti proliferation effects of four different concentrations of tea polyphenols on PG cell lines were determined with MTT assay. Laser scanning confocal mircroscopy and flow cytometry techniques were used to determine the changes in intracellular calcium concentration, GJIC, Cx43 expression, and cell cycle distribution after treatment with tea polyphenols. The results showed that four different concentrations dose dependently inhibited the proliferation of PG cell lines. It caused a decline in the proportion of cells in S and G2/M phase, blocked the cell cycle progression in G0/G1 phase, and reduced the proliferation index of PG cell lines. Compared with control group, intracellular calcium concentration, GJIC and Cx43 expression were gradually increased ascended with an increase in tea polyphenols concentration. The results suggested that tea polyphenol could inhibit growth of PG cell lines. The mechanism of anti tumor is associated with an up regulation of GJIC of PG cell line.