Molecular Cloning of Protective Antigen Toxin Gene of Bacillus anthracis Sterne Strain 34F2 Sterne Strain in pET28a Vector

Anthrax is endemic in various pockets of India, but many cases remain undocumented due to lack of a proper diagnostic system. Anthrax is caused by Bacillus anthracis, a Gram-positive, spore forming, rod-shaped bacterium The clone construction has remained a very essential step in any expression study. The protective antigen toxin of the B. anthracis is the central toxin and extensively studied for its applicability in diagnosis and prophylaxis. This study reports construction of a PA-pET28a clone which will act as a backbone for expression study and analysis. Full length PA gene was amplified using the genome of Sterne vaccine strain as template. The complete PA gene (~2226bp) was amplified after optimizing the PCR annealing conditions using gradient PCR followed by RE digestion of the insert and vector. The digested vector and insert were ligated and transformed in to E. coli TOP 10 cells for screening and isolation of the PA-pET28a clone. The in-silico characterization of the PA protein indicated the soluble nature and high antigenicity of the protein. The PA-pET28a clone will serve as a backbone for expression studies and development of a candidate for suitable diagnostic and vaccines in livestock.