Identification, cloning, sequencing virG gene and construction a live Attenuated ΔvirG(icsA) strain byusing λ Red recombinase

Shigella and enteroinvasive Escherichia coli (EIEC) strains are belong to the gram negative bacterium family which cause the most communicable of bacterial dysenteries (shigellosis). The virG gene as an essential factor is required for pathogenicity of shigella. This protein encoded via virG gene which located on the outer membrane (OM) of bacteria surface. Furthermore، the VirG protein belonging to autotransporter (AT) protein family of extracellular protein''s gram-negative bacteria. This protein interacts with the host actin regulatory protein which caused intra- and intercellular spreading throughout the host epithelium. One approach for construction of Shigella vaccines is to attenuate wild-type strains by mutating genes which regulate specific virulence properties. The aim of this research was identification، cloning، sequencing virG gene and construction of a live attenuated ΔvirG by use of the λ Red recombinase in Shigella dysenteriae type 1isolated from patients with shigellosis. By use of serological and Polymerase Chain Reaction (PCR) tests، species and serotype of shigella seperated from patient was confirmed. According to the Gene Bank database (NCBI)، detection primers of virG gene was designed، after amplification of virG، this construct was cloned to pGEM-7zf vector as cloning vector. Finally، sequencing was performed by use of universal primers of vector. The pKD46 as helper plasmid (composed of the essential genes for induce recombination under arabinose promoter) was transformed to the native shigella by using electroporation. After designing the primes which require for induction recombination، the PCR reaction performed through pKD3 vector (which compose of chloramphenicol cassette flanked by FRT sequence). After purification of chloramphenicol cassette، this cassette transformed to the native shigella which carrying pKD46 by using electronic shock. Mutant strain obtained by occurring homologous recombination between chloramphenicol cassette and virG gene. Then، antibiotic cassette was eliminated via FRT sites by application of pCP20 (carrying FLP recombinase) as helper plasmid. Precision of process was confirmed by phenotypic (growth resistant strain to chlramphenicol) and genotypic (PCR reaction with external primers and then sequencing of its product) characterization. The result sequencing of virG in comparison with standard strain as reference strain (Accession No. CP000035) was identical (100%). According to bioinformatics analysis mutant strain، deletion of 3220 bp of virG gene was confirmed. Utilization of λ Red recombinase system facilitates mutant construction in more cost-effective method in comparison with the other techniques such as suicide vector.