Identification, isolation and sequencing of PGA gene from shigella boydi

Introduction and objectives Biology is a rich science regarding advances in experimental techniques that produce various proteins and other materials. The resulting product, 6-aminopenicillanic acid (6-APA), is used for the production of a variety of semi synthetic Penicillin. Formation of active PGA includes a series of post translational steps via translocation and periplasmic processing. The production of PGA by recombinant protein technology can produce PGA enzyme more and cheap. The goals were: 1. screening of non E.coli members of Enterobacteriaceae from environmental and clinical specimens for PGA by PCR. 2. PGA cloning from non E.coli bacteria and sequencing of the gene. Materials and Methods In this study, 290 non 15TE. coli15T, Enterobacteriaceae were isolated from environmental and clinical specimen. One PGA positive strain (Shigella boydii) was isolated. DNA was separated from this isolate and 15Twas15T entered in PCR reactions using primers designed on conserved region of PGA genes. The PCR product was cloned in pGEMTeasy vector. Results Sequencing of cloned portion indicated that the gene encoding Penicillin G acylase from Shigella boydii included an open reading frame of 2538nucleotide encoding 846 amino acids. Survey of sequencing result revealed that this gene contain 98% homology to previously reported PGA from Shigella boydii strains. Conclusion Results of this study showed that the polymerase chain reaction method is a very sensitive and specific method that can be utilized for screening of PGA genes from different samples. Furthermore, it is possible the application of pathogenic bacteria as a resource of essential genes.

کلیدواژه ها:

PGA ، PCR ، Shigella boydii ، identification ، sequencing