Optimizing factors for the efficient expression and purification of spao and lamb from Salmonella typhi

We optimized the expression and purification of outer membrane proteins SpaO and LamB from Salmonella typhi. We investigated various factors in the expression and purification processes, including the use of isopropyl ß-d-1 thiogalactopyranoside (IPTG), imidazole, and urea. First, PCR amplification was carried out on SpaO and LamB genes. The genes were then cloned in pTZ57R/T, and then expressed in pET28a vector and transformed into Escherichia coli BL21 (DE3). Gene insertion was confirmed by enzymatic digestion with NdeI and XhoI. Inclusion bodies expressing recombinant SpaO and LamB were induced with 200 and 400 µL 0.5 mM IPTG, respectively. The formed protein inclusion bodies were then isolated from the pellet and solubilized in IB buffer containing 8 M urea for SpaO and 6 M urea for LamB. Proteins were refolded by dialysis in 3M urea. Purified proteins with nickel-nitrilotriacetic acid affinity chromatography and eluted with buffer containing 250 mM imidazole for SpaO and 150 mM imidazole for LamB. The protein expression profiles were analyzed by SDS-PAGE, which identified the 33 and 49 kDa bands corresponding to rSpaO and rLamB. Western blotting Purification was carried out by nickel affinity resin with 250 mM and 150 mM imidazole for rSpaO and rLamB and refolded through stepwise dialysis with anti-His tag antibodies confirmed their expression. These optimized methods can be used to generate recombinant proteins for the development of future vaccines.

Anti-hyperlipidemic and anti-diabetic evaluation of ethanolic leaf extract of Catharanthus roseus alone and in combination therapy

The use of plants in disease treatment is cost effective and relatively safe. This study was designed to investigate anti-hyperlipidemic and anti-diabetic activity of ethanolic leaf extract of Catharanthus roseus alone and in combination therapy in hyperlipidemic & diabetic mice. Eight groups comprising five mice each were used. Group A was hyperlipidemic control, group B, C, D received atorvastatin (20 mg/kg), leaf extract (200 mg/kg) and leaf extract in combination with atorvastatin (200 mg/kg and 20 mg/kg) orally for 15 days. Group E was diabetic control. Group F, G, H received sitagliptin (40 mg/kg), leaf extract (200 mg/kg) and extract in combination with sitagliptin (200 mg/kg and 40 mg/kg) orally for 7 days. Blood cholesterol levels were measured at 1st, 5th, 10th and 15th day and fasting blood sugar levels were measured at 2, 12, 24, 72 and 168 hours during treatment. One-way ANOVA with tukey- kramer multiple comparison test was used. The chemical characterization of ethanolic extract of Catharanthus roseus leaves showed presence of alkaloids, saponins, tannins and flavonoids. Ethanolic extract of Catharanthus roseus has significant anti-hyperlipidemic & anti-diabetic effects (p<0.05, p<0.01) when compared with control but had not cause significantly increase in anti-hyperlipidemic effects of atorvastatin. While significantly increased the antidiabetic effect of sitagliptin (p<0.05)