Preparation And Immunomodulating Function Of Klebsiella Pneumoniae Capsule Glycoprotein

The indirect enzyme-linked immunosorbent assay (ELISA), the fermentation conditions, the extraction methods and the immunocompetence of Klebsiella pneumoniae (Kp) capsule glycoprotein were involved. The main research contents and results were as follows:A indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect and quantitate the Klebsiella pneumoniae and its capsule glycoprotein. The specific and high titer rabbit antiserum of Kp capsule glycoprotein was prepared successfully using a purified Kp glycoprotein as antigen and was purified with ammonium sulfate fractionation and adsorption. The sensitivity of the assay is 0.031 g/ml. The standard curve is linear from 2.5 g/ml to 30 g/ml. The error of inter-plate is from 1.04% to 3.22%, and the error of infra-plate is from 2.61 % to 8.35%.The results of single factor and orthogonal experiment showed that the optimized medium composition contained: sucrose 4%, tryptone 2%, yeast infusion (powder) 0.1%, K2SO4 0.1%, MgSO4 0.025%, KH2PO4 0.4%, Na2HPO4 0.3%. The optimized fermentation conditions in shake flask were as follows: inoculum age 5h, inoculum size 2%, temperature 37 , volume of medium 16%, shaker speed 150r/min, pH6.0, fermentation time 3.5h. The yield of shake flask fermentation was 1.9 1010cfu/ml, and it was improved to 2.4 1010cfu/ml in batch culture.The extraction methods of Kp capsule glycoprotein was studied. The bacteria was lysed by trypsin, NP40 and lysozyme. The lysate was concentrated by ultrafiltration on 50kDa molecular filter membrane and was extracted 1h by methanol and dimethoxymethane ( 1:4 ) 5 times of volume under agitation. After deproteination, precipitation and drying, the yield of rough extracted preparation was 2.02g/L, containing 10.2% available component. Then the extracted preparation was purified by ion exchange chromatography and gel exclution chromatography. The final yield of Kp capsule glycoprotein was 0.151g/L, containing 81.3% available component.The extracted preparation and Biostim were determined and compared by HPLC. Onthe performed HPLC chromatographiccondition the extracted preparation showed only one peak at the same time as Biostim.The effect of the extracted preparation and Biostim on splenocyte proliferation responses induced by ConA were assayed by MTT colorimetic method in vitro. It was founded that the glycoprotein of the extracted preparation, as well as Biostim responsed to splenocytes in a dose-dependent manner, and sharply enhanced the immunostimulative function compared with the control. The extracted preparation was more powerful than Biostim.