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Pakistan Journal of Pharmaceutical Sciences. 2014; 27 (6): 1905-1910
in English | IMEMR | ID: emr-148836


The inhibition of 3-hydroxy-3-methylglutaryl CoA reductase [HMGCR] is considered able to decrease serum cholesterol levels and dramatically reduce the risk for cardiovascular and cerebrovascular diseases. The statins, competitive inhibitors of HMGCR, have been employed to control hypercholesterolemia. But their side effects, especially their safety of long-term administration have attracted great attention. Therefore, there is still an urgent requirement for the development of safer inhibitors of HMGCR with less serious side effects. In this study, we cloned and purified the catalytic domain of human HMGCR [delta HMGCR], and applied the method of Ultra Performance Liquid Chromatography [UPLC] to assay delta HMGCR activity and screen its inhibitors from natural products. The results indicated that EGCG can inhibit delta HMGCR in the presence of some glycerol in vitro and can decrease cellular total cholesterol in HepG2 cells. As a consequence, it is promising to put EGCG into the development of hypolipidemic health product

Hydroxymethylglutaryl CoA Reductases , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Glycerol , Chromatography, High Pressure Liquid
Chinese Journal of Biotechnology ; (12): 216-219, 2005.
Article in Chinese | WPRIM | ID: wpr-249922


The lysin gene of Bacillus anthracis-diagnosing bacteriophage, obtained by PCR amplification,was cloned into the Escherichia coli exepression vector pET22b which has been cleaved by EcoR I and Nde I. The recombinant vector pET22b-gamma lysin was verified to be correctly constructed by PCR, sequencing and enzyme digestion, and highly expressed in E. coli BL21 (DE3), which accounted for about 40 percent of total protein in E. coli BL21 (DE3), while in the 5L fermentor the expression level reached 15g/L. After expression, disruption and purification with three-step chromatography, Streamline SP, SP HP and Sephacryl S-100, the recombinant gamma lysin was finally obtained with purity of higher than 95 percent as determined by gel scan. The final yield following SP HP was 19.1 percent, with a greater-than-350-fold increase in specific activity. The pure enzyme has been shown active to Bacillus anthracis, and not to E. coli, Bacillus subtilis and Bacillus cereus. Its specific activity was about 1400 u/mg.

Bacillus anthracis , Virology , Bacteriophages , Cloning, Molecular , Escherichia coli , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Metabolism , Viral Proteins , Genetics
Chinese Journal of Biotechnology ; (12): 667-672, 2004.
Article in Chinese | WPRIM | ID: wpr-249957


The aim of this study is to construct a phage display single-chain variable fragment (scFv) library against breast cancer cells and screen the specific antibodies against MCF-7 cells from the library. The BALB/C mice were immunized with MCF-7 cells. Total RNA of spleens was isolated. The heavy-chain (VH) and light-chain variable region genes (VL) of the antibodies were amplified by RT-PCR and joined into a single chain by overlapping PCR with a linker DNA encoding the peptide (Gly4Ser)3. The assembled scFv fragments were cloned into the phagemids(pCANTAB5E) and the recombinant phagemids were used to transform competent E. coli TG1. The transformed TG1 cells were infected by helper phage M13KO7 and the recombinant phagemids were rescued. The scFv fusion proteins were displayed on the surfaces of the recombinant phages. A phage display antibody library of repertoire of 1.2 x 10(6) clones was constructed. The specific antibodies against MCF-7 cells were enriched by 75 times after five rounds of affinity selection. Ten recombinant phages clones that exhibited specific binding to MCF-7 cells were identified. The specificity of those phage clones was analyzed by reactivity against HepG2 cells and Hela cells by ELISA. One of the selected phage clones against MCF-7cells was used to infect E. coli TOP10 to produce the soluble scFv antibodies after induction with IPTG. The strategy of construction and screening of antibody library directed against the whole tumor cells described in this report should be generally applicable to generate tumor cell-specific antibodies.

Animals , Female , Humans , Mice , Antibodies, Neoplasm , Genetics , Allergy and Immunology , Breast Neoplasms , Allergy and Immunology , Therapeutics , Cell Line, Tumor , HeLa Cells , Hep G2 Cells , Immunoglobulin Heavy Chains , Genetics , Immunoglobulin Light Chains , Genetics , Immunoglobulin Variable Region , Genetics , Mice, Inbred BALB C , Peptide Library , Single-Chain Antibodies , Genetics , Allergy and Immunology