Cloning, sequencing, and in silico characterization of Omp 28 of Salmonella Typhi (strain MTCC 733) to develop r-DNA vaccine for typhoid fever

Abstract

Background:Typhoid is one of the most important diseases of human beings caused by Salmonella Typhi. There are many vaccine reported against Salmonella Typhi, but search for new candidate vaccine antigens is still going on because presently available vaccines have several limitations such as short-term immunity, high cost, and allergic reaction. Several approaches such as subunit vaccines, Vi polysaccharide, mutant vaccines, and r-DNA vaccines have been tested. r- DNA vaccines have shown some promising potential (targeted Omp). Omp 28 had shown very promising results and suggests that it should be used in further studies of animal protection against the disease.Objective:Cloning, Sequencing and In silico analysis of Omp 28 gene to develop r-DNA vaccine of S. Typhi.Materials and Methods:Omp 28 is made up of three identical subunits of 9.6 kDa showing PCR amplicon of 330 bp which has been cloned in the pJET vector. Recombinant clones has been sequenced, and data submitted to NCBI. Secondary structure was deduced by the Chou Fasman and Garnier method. The sequence of Omp 28 was studied for antigenic indexing, epitope mapping, and MHC mapping using various bioinformatics tool.Results and Conclusion:The sequence of Omp 28 has been assigned accession no GQ 907044.1 by NCBI. Secondary structure has shown it has more alpha region. Hydrophobic plot and surface probability plot shows most amino acids are surface exposed which is a requirement to develop a r-DNA vaccine. Antigenic sites are located within surface exposed regions and eight antigenic determinants are present in Omp 28. On Prosite analysis of Protein shown two motifs i.e. anaphylatoxin domain signature motif at position 219-252 and other one was iron sulphur binding region signature motif at position 36-44. On epitope analysis total six major B cell epitopes were observed which can provoke humoral immunity. On T cell epitope mapping several major epitopes has been found in case of MHC class I and MHC class II. It indicates that Omp 28 can provoke cell mediated as well as humoral immunity and can be proven a promising candidates of Salmonella Typhi.

Cloning, expression and purification of the outer membrane protein28 of Salmonella enterica serovar Typhimurium for subunit vaccine development - a short communication

“…Upon amplification the size of the Omp28 gene from S. Typhimurium was found to be 330bp M. Pandey et al: Cloning and epression of Omp28 of S. Typhimurium (Fig. 1), which was similar to that observed by SAXENA et al (2012) for the Omp28 gene of S. Typhi. The amplified product was then cloned in pJET1.2/blunt end cloning vector and transformed in to DH5α cells.…”

“…The outer membrane proteins (Omps) from Salmonella have been proven to have good immunogenicity (LEE et al, 2010;GIL-CRUZ et al, 2009). Since that discovery, Salmonella Omps have been characterized (HAMID and JAIN, 2008) and targeted for protection (JHA et al, 2015;SAXENA et al, 2012;BHAT and JAIN, 2010). Omp28 was isolated independently from S. Typhi by BHATNAGAR et al (1982) and DE ANDRADE et al (1998).…”

Cloning, Sequencing and In Silico Characterization of OmpF Protein of Salmonella typhimurium for its Immune-Potential

“…Phenylalanine at C terminus provide stability and proper assembly of protein into the outer membrane (Ruffolo et al, 1996) Surface proteins of Salmonella have been considered as a potential vaccine candidate. Some of the outer membrane protein had been studied and described its immunogenic potential (Jha et al, 2012;Saxena et al, 2012;Jha et al, 2015).To date, there are only limited studies were conducted on OmpF protein of Salmonella typhimurium. The functional unit of OmpF porin is a homo-trimer and is formed by 16-stranded beta-barrel with three beta barrel monomers in its outer membrane (Balasubramaniam et al, 2012).…”

Boğmaca hastalığına karşı yeni aşı tasarımı ve analizi

“…[7] Rekombinant aşıların üretime geçmesinden önce in silico ortamda aşının tasarlanması ve farklı programlarla analiz edilmesi gerekir. [8] Yeni nesil aşılar Rekombinant DNA Teknolojisi ile elde edilerek daha güvenilir olmaları ve uzun süre insanları koruyabilmeleriyle ilgili araştırmalar vardır. [9][10][11][12] In silico (Bilgisayar Ortamı) çalışmaları aşı keşfinin önemli bir parçası haline gelmiştir.…”