[Cloning virG gene and generation mutant construct of pGEM delta virG in order to induction recombination in native Shigella dysenteriae]

ABSTRACT

Shigellosis disease is the major causes of morbidity in children with diarrhea in Iran. The virG [icsA] gene plays a key role in pathogenesis and ability of invasion in shigella. The aim of this study was cloning, sequencing virG gene and developing a mutant construct pGEM delta virG in order to induction recombination in a native shigella for generation a live attenuated vaccine candidate strain. Initially, by use of biochemical tests, the native shigella strain was detected. The virG gene was cloned in pGEM-7zf vector and the nucleotide sequence was determined. According to the data of sequencing, digestion mapping of pGEMvirG vactor was obtained and a part of virG gene by using enzymatic digestion was removed. Finally, pGEM delta virG construct was transformed to E. coli by utilization of chemical transformation method. The native shigella strain by using biochemical tests was confirmed. The result of sequencing virG gene [native strain] was submitted in NCBI Genebank database. The pGEM delta virG construct contains a mutant construct of virG gene which 1751 bp was deleted through enzymatic digestion reaction and transformed in E. coli. Using the technique of allelic exchange based on the incident of recombination in bacteria is one of the most effective methods to develop a disruption in the target genes. This mutant construct can be applied in development of a live attenuated Shigella dysenteriae vaccine candidate.