The preparation and identification of diagnostic recombinant glycerol kinase / 生物医学工程学杂志

In order to establish an efficient and low-cost production procedure of recombinant glycerol kinase (r-GK), we expressed the r-GK gene at high level in E. coli by induction with lactose on a large-scale fermentation of 300L. The results showed that the biomass concentration reached OD600 of 42 and the expression of r-GK in E. coli accounted for about 30% of total soluble protein. The cell-free extract was processed by selective thermo-denaturation and then purified with Ni sepharose FF column chromatography. Finally, highly purified r-GK was obtained and its purity reached 97% by using analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), polyacrylamide gel electrophoresis (PAGE) and gradient polyacrylamide gel electrophoresis (Gradient PAGE). Further identification study showed that the molecular weight of r-GK was 120kDa with two subunit of 58kDa. Contaminants of NADH oxidase and catalase were not detected in the sample pool of r-GK. The purified r-GK was able to retain about 85% of its initial activity at 4 degrees C for 30 days. After lyophilized, it can retain 93% of its initial activity at 4 degrees C for one year.

Preparation and identification of monoclonal antibody against alpha-momorcharins / 生物医学工程学杂志

Three BALB/c mice were immunized four times with alpha-momorcharins (alpha-MMC). Using polyethylene glycol (PEG) method, the immunized splenocytes were fused with SP2/0 cells. One strain of hybridoma cells was obtained which secrete antibodies against alpha-MMC. To get ascites, the hybridoma cells were injected into the abdominal cavity of mice. The antibodies were purified from ascites. Indirect enzyme linked immunosorbent assay (ELISA) and Western blot assay were applied to determine the specifity of the monoclonal antibody (McAb). The results showed that the McAb was specific to alpha-MMC without detectable cross-activity with MAP30. The McAb provided detecting method for further research of the structure and function of alpha-MMC.

Preparation of 5-fluorouracil-peanut agglutinin conjugate and determination of the ratio of drug to conjugate / 生物医学工程学杂志

5-fluorouracil was combined with peanut agglutinin by a water-soluble carbodiimide to prepare the tumor target conjugate of 5-fluorouracil-peanut agglutinin and the ratio of drug to conjugate was determined by the modified trinitrobenzenesulfonic acid method (TNBS). The ratio of drug to conjugate was 76.33%. The result showed that 5-fluorouracil could link to the peanut agglutinin by EDC/NHS crosslinking with high drug ratio.

Inhibition of proprotein convertases by mung bean trypsin inhibitor / 第二军医大学学报

Objective:To extract the mung bean trypsin inhibitor(MBTI) from mung bean and to study the inhibitory activity of MBTI against proprotein convertases(PCs).Methods: MBTI was purified to homogeneity by ammonium sulfate precipitation, sequential chromatography of gel filtration,ion exchange,affinity chromatography and HPLC.The high expression strains of 2 PCs,Kexin and Furin,were selected.Kexin and Furin were purified by ammonium sulfate precipitation and gel filtration.The inhibitory activity of MBTI against Kexin and Furin was assayed and the inhibitory constants(Ki) of MBTI against the 2 PCs were calculated by Dixon's plot.Results:The purified MBTI showed a single peak on HPLC and a single band on SDS-PAGE.The inhibitory activity of MBTI against Kexin(Ki= 3.9?10~(-9)mol/L) was stronger than that against Furin.Conclusion: MBTI can inhibit both Kexin and Furin,especially Kexin,and also can be an ideal inhibitor against PCs after further modification.