Cloning of the glyceraldehydes 3-phosphate dehydrogenase gene of porphyromonas gingivalis and its expression in E. coli / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To clone the glyceraldehydes 3-phosphate dehydrogenase (GAPDH) gene of Porphyromonas gingivalis (P. gingivalis) and to induce its fusion expression in E. coli.</p><p><b>METHODS</b>GAPDH was obtained by PCR and was inserted into cloning vector pMD-18-T to construct clone recon. Double enzymes digest the recon pMD18-T-GAPDH and the prokaryotic expression vector pET-32a and then connect to get the expressing recon pET-32a-GAPDH. The recombinant expression plasmid which had been confirmed by enzymes digestion was transformed to E. coli competent cells BL21 and induced the expression of GAPDH with isopropyl beta-D-1-thiogalactopyranoside (IPTG) of different density.</p><p><b>RESULTS</b>DNA sequencing showed that the fragment was 99.802% the same to the sequence published in NCBI. Under the best density, IPTG could be highly expressed.</p><p><b>CONCLUSION</b>The GAPDH had been successfully cloned and expressed in E. coli which gets ready for the following experiment to study the immunity of GAPDH and the homologues antibody preparation.</p>

Antioxidative and aldose reductase-inhibitory effects of a fermentation filtrate of Rubus coreanus / 한국실험동물학회지

Antioxidative and aldose reductase (AR)-inhibitory effects of a fermentation filtrate of Rubus coreanus (FRC) were investigated using corneal/retinal homogenate and lens cytosol, respectively. Rat corneal/retinal homogenate was treated with 50 microM FeCl3 in the presence of FRC (3.2-100 microg/mL) for 30 min at 37degrees C, and thiobarbituric acid-reactive substances (TBARS) was quantified as a lipid peroxidation parameter. FRC markedly suppressed the TBARS production in a concentration-dependent manner, leading to 50% (IC50) and 100% (IC100) inhibitory concentrations of 20 and 95 microg/mL, respectively, which was similar to the effect of butylated hydroxyanisole. Activity of AR from rat lens was assayed in the presence of FRC (1-31.6 microg/mL) at 25degrees C using glyceraldehyde as a substrate. FRC inhibited lens AR by 50% (IC50) and 90% (IC90) at approximately 2 and 31.6 microg/mL, respectively, comparable to the effect of quercetin. The results indicate that ERC could be a promising candidate for the improvement of eye injury and visual dysfunction of dry eye and diabetic patients.

Cloning and expression of Lactobaceillus reuteri glycerol dehydratase gene in Escherichia coil / 生物工程学报

There is growing interest in biodiesel and this results in the accumulation of glycerol. The exploitation and application of glycerol has attracted more and more attention. In the current study, glycerol was biotransformed to produce 3-hydroxypropionaldehyde by genetic engineering bacteria. It is known that 3-hydroxypopionaldehyde has been widely used as an important intermediate for chemicals, effective antimicrobial agent, and fix agent for tissues. A pair of primers was designed on the basis of the sequence of both NH2-terminus and the amino acid sequence of glycerol dehydratase reported by NCBI, and a fragment about 1.6 kb was obtained by PCR amplification using the total genome DNA of Lactobacillus reuteri as template, then the fragment was cloned to the pMD18-T vector and sequenced. Two specific primers were designed according to the obtained sequence, and a fragment with length of 1674 bp was amplified using PCR with these two specific primers. Consequently, the resulting products were digested with EcoR I and Hind III and ligated using T4 DNA ligase to the pET28b vector digested with the same enzymes. The recombinant plasmid, named pET28b-dhaB, was transformed into E. coli BL21. The positive clones were induced with IPTG and the expression products were further analyzed by SDS-PAGE, indicating that protein with a molecule weight of around 65 kD was obtained. Furthermore, the glycerol dehydratase activity was evaluated and compared with the wild type strain as well.