Studies On Extraction And Purification Of The Capsular Glycoprotein From Klebsiella Pneumoniae And Its Immunomodulatory Effects

The capsular glycoprotein from Klebsiella pneumoniae (Kp) is main ingredient of Biostim , which is an immonumodulating agent produced by Roussel Corporation, France. On the basis of other researchers, the crude product of Kp capsular glycoprotein was extracted from Kp-9 strain isolated clinically in China after bacteria lysis, concentration, delipidation, deproteination and precipitation. Three times of above extraction indicated that such method had satisfying stability and reproducibility.The crude product was treated with Cetyl trimethyl ammonium bromide (CTAB) in order to separate the neutral glycoprotein and the acidic glycoprotein. The optimal condition according to ELISA result was: pHV.O, 1% of crude product and 3% of CTAB. The result of immune blot test suggested the neutral glycoprotein was the active part of crude product and its purity was 53.75% rised from 29.99% of origin. The crude product and neutral glycoprotein were further purified by RP-HPLC and their purify increased to 93.4% and 96.3% respectively. The main composition of purified product (33.0% of protein, 24.7% of sugar, 4.1% of glucuronic acid) was similar to those of Biostim (30-45% of protein, 30-45% of sugar, <4% of glucuronic acid), though the content of sugar was slightly lower. Molecular weight of the purified product determined by high performance gel filter chromatography was 60634D, which was approximately equivalent to that of Biostim (55036D).The immunomodulatory effects of crude product were detected by lymphocyte transformation test (LIT) and plaque forming cell assay (PFC). It was found that all four groups (lOug/ml, 20|ag/rnl, 40ug/ml, 80u?ml) could activate proliferation of T lymphocyte at different levels and 20u.g/ml group had the strongest stimulation. PFU result indicated that 20ug crude product promoted antibody forming of murine B lymphocyte compared with the control (P<0.01). There was no statistically difference between the number of plaques formed by crude product and Biostim (P>0.05).In addition, we observed the protective effects of crude product of Kp capsular glycoprotein on mice infected by Escherichia coli (E. coli) orStaphylococcus aureus (S. aureus). Three groups of mice were injected with crude product at the dose of 3mg/kg (high dose group ) and 5mg/kg (low dose group ) and normal saline respectively. Five days later, in the E.coli group the survival rate of high dose group was 87.5% compared with 12.5% of the control (P<0.05). In the S. aureus group the survival rate of low dose group was 87.5% and 100% of high dose group, and these numbers were higher than that of the control (PO.05 of the former, PO.01 of the latter).The average antibody titer of the immune mice was assayed by ELISA method. In the E.coli group the antibody titer of low dose group was 1:443 and 1:508 of high dose group. In the S. aureus group the values were 1:408 and 1:497 respectively. Compared with the control, the average antibody titer of experiment group was significantly higher (P<0.01).This research work reconfirmed the practical effects of Kp-9 strain. The technics and conditions of purifying Kp capsular glycoprotein at laboratory level were established and the immunomodulatory effects in vitro and in vivo were studied. These results laid preliminary basis for the nationalization of Biostim.