Study On The Capsular Immunocompetent Agent Of Klebsiella Pneumoniae

Klebsiella pneumoniae CGMCC No. 1000 strain,which was screened from clinic, has the typical characteristic of serum type infected by Klebsiella pneumoniae. The capsular immunocompetent agent (CIA) of Kp CGMCC No. 1000 was a potential immunomodulater,the fermentation and preparation process and immunoactivity of CIA were studied systematically. Capsular polysaccharide(CPS), one of component of CIA, was then purified, it's capability to induce the K562 tumor cell apoptosis and the basic structure was investigated in succession.In order to investigate the CIA, an Indirect Enzyme — Linked Immunosorbent Assay(ELISA) of CIA was founded, with sensitivity of 0. 031 μ g/mL, valid detective range of 2.5 μ g/mL～30. 0 μ g/mL. An Indirect Cell ELISA was established and investigated to evaluate the fermentation process of Klebsiella pneumoniae, it showed a good correlation to the yield of CIA, both indirect cell ELISA and biomass were utilized to evaluate the fermentation process.The influence of substrate composition and growth conditions on CIA expressed by Klebsiella pneumoniae using submerged cultivation were investigated. The statistical optimization of media and growth conditions in shake flask were established. The biomass and activity of the culture under the optimum process were enhanced by 72.7 and 33 times respectively, then the process was magnified and improved in stired tank fermenter.The lysis of cell is the first key step in the preparation of CIA, under the co-action of trypsin, lysozyme and NP-40, the cell lysed within 2h,then the lysate was concentrated by ultrafiltration which serves as concentrating and partial purifying action simultaneously.Crude CIA was de-nucleic acid through CTAB-NaCl precipitation, then CIA was got by ethanol preparation.Many tests were employed to evaluate the immunoactivity of CIA.The results show that CIA can enhance T lymphacytes proliferation and quantity of antibody-forming B lymphocytes, can induce the cytokine TNF production in a certain degree. Also CIA can increase MO activity which has been proved in both normal and immunodepressedanimals.CPS was purified successively through the CTAB precipitation, Ion-exchange and gel filtration, the homogeneity of CPS was judged by the gel filtration and agarose gel electrophoresis, acting as immunogen, CPS produced high titer and strong affinity polyclonal antibody. The effect of CPS on the cell immunoactivity was also studied, the results show that CPS possesses immunoregulation on the spleen cells of mice significantly.Apoptosis-resistant chronic myelomonocytic leukemia K562 cells was selected to study the cell-type-specific characteristics of cell growth inhabition and apoptosis induced by CPS. The results show that CPS can inhibit the growth of K562 cells, arose the cell cycle changing and cell apoptosis according to the CPS concentration and reaction time. It seems there is some relationship between cell cycle changing and the degree of K562 cell apoptosis.Structure of the CPS from Klebsiella pneumoniae CGMCC No. 1000 strain has been determined by many physical and chemical techniques. Molecular weight of CPS is about 109194.3, it is composed of heavy saccharide chain with peptide and a little nucleic acid. Monosaccharide composition is ribose> mannose> glucose> galactose and hexuronic acid. The peptide chain is composed of 17 types of amino acid. ￡ -elimination, periodate oxidation and NMR spectroscopy establish the nature of anomeric linkages.There exists a - and 3 - anomeric linkages, 1 — 6-linkage between the monosaccharides. and O-glycosidic linkage between the monosaccharide and peptide chain.