Cloning and periplasmic soluble expression of hepatitis B surface antigen gene in Escherichia coli.

OBJECTIVE: The objective of this study was to clone and express the hepatitis B surface antigen gene (HBsAg) in Escherichia coli (E. coli), thereby aiming to develop potential local therapeutics for combating Hepatitis B virus (HBV) infection in the Pakistani community by producing HBsAg in E. coli. MATERIALS AND METHODS: Blood serum samples were collected from hepatitis B-infected patients, and their genomic DNA was extracted. Real-time and nested polymerase chain reaction (PCR) was performed to amplify the HBsAg gene. The gene of interest was cloned into the pET20b expression vector and transformed into E. coli BL21 (DE3) using Isopropyl ß-D-1-thiogalactopyranoside (IPTG) induction. The gene's precise size was confirmed with gene-specific external and internal primers (681 bp and 400 bp, respectively). RESULTS: The HBsAg gene was successfully sequenced and submitted to GenBank, exhibiting 98% homology with targeted HBV sequences worldwide. The expression of HBsAg protein was confirmed through silver staining, Coomassie staining, western blot, and dot blot analysis. CONCLUSIONS: The expressed protein clones are now available for further development as a local recombinant DNA vaccine to prevent hepatitis B viral infection in the local community.

Brassica oleracea L. (Acephala Group) based zinc oxide nanoparticles and their efficacy as antibacterial agent.

Zinc oxide nanoparticles were synthesized from the leaf extract of Brassica oleracea L. Acephala group (collard green) followed by their characterization using Scanning Electron Microscope (SEM), and Energy Dispersive X-ray (EDX). The antibacterial properties of zinc nanoparticles were tested against Gram-negative bacteria, Pseudomonas aeruginosa (ATCC ® 9027™), Escherichia coli (ATCC ® 8739™), Klebsiella pneumoniae (ATCC® BAA-1705™) and Gram-positive bacteria, Staphylococcus aureus (ATCC ® 6538™) and Listeria monocytogenes (ATCC ® 13932™), at four different concentrations (50.00 µg/ml, 100.00 µg/ml, 500.00 µg/ml and 1 mg/ml) of zinc oxide nanoparticles suspension. Results revealed that the synthesized nanoparticles exhibit strong antibacterial effects against Pseudomonas aeruginosa, Listeria monocytogenes, Klebsiella pneumonia, Staphylococcus aureus and Escherichia coli at 500.00 µg/ml-1 mg/ml concentrations. An increase in efficacy of nanoparticles with the decrease of their size was also evident. This is a first ever report on Brassica oleracea, L. based nanoparticles which demonstrates that 500.00 µg-1 mg/ml conc. of zinc oxide nanoparticles have antibacterial activity against both Gram -ve and Gram +ve bacteria and have the potential to be considered as an antibacterial agent in future.

Identification of hepatitis B virus core mutants by PCR-RFLP in chronic hepatitis B patients from Punjab, Pakistan.

Chronic hepatitis B virus (HBV) infection remains a major health issue worldwide. Several factors including core gene variation are responsible for the development of chronicity of HBV infection. The present study was designed to identify the variations in the core region of the HBV genome in a local population of chronic hepatitis B patients (n = 57) using a PCR-based restriction fragment length polymorphism (PCR-RFLP) method. Fifty subjects were found to be positive for the presence of HBV DNA. For the core region genotyping, the Ava II and Msp I restriction enzymes were used. Mutations at nucleotide (nt) 2147 and nt 2362 in the HBV genome in the core region for Ava II (A4 type, 74%) and nt 2331 for Msp I (M1 type, 66%) were observed as the most common pattern. These results are different from those of previously reported studies on other populations and thus appear to be unique to the Pakistani population. This type of characterization of core mutants may be useful for the design of vaccines based on viral epitopes that are effective for the Pakistani population. Moreover, these unique genotypic patterns for the HBV core gene might be some of the main factors responsible for understanding the underlying mechanism by which HBV chronicity is developed in the Pakistani population.