Bioinformatic Design and Construction of a Chimeric Gene Comprising the stxB, ctxB and blf1 Genes

Authors
zahra moshiri 1
raheem haddad 1
hosein honari 2
1 Department of Biotechnology, Imam Khomeini International University, Qazvin, Iran
2 Biological Research Center, Imam Hossein Comprehensive University. Tehran, Iran
Abstract
Introduction

bacteria species; Burkholderia pseudomallei, Vibrio cholera, and Shigella dysenteriae are the main pathogen causing Melioidosis, Cholera, and Shigella in humans respectively. bacterial species have a protein that has antigenic prosperities along with being a mucus based adjuvant thus easing their delivery and immunogenicity. These proteins were BLF1(B. pseudomallei), CTxB (V. cholera), and StxB (S. dysenteriae). The goal of this study was a chimeric gene composed of Blf1, CTxB, and StxB and expression of these proteins from E.coli.

Materials and Methods

Using Bioinfoamtic tools such as Protparam and Modeller the different combinations of the chimeric protein were assessed. The blf1 and stxB genes were PCR amplified from pET28a-BFL1-StxB. The 850bp fragment encompassing BFL1-StxB was cloned using SalI and NotI in the pET28a-CTxB vector. The engineered plasmid was transformed in BL21(DE3). The ctxB-stxB-blf1 gene was expressed following induction with IPTG. This induction was then assessed using SDS-PAGE.

Results

Chimeric protein instability index gets 40.06 and the half-life of it in E. coli was over 10 hours. Codon Adaptive Index (CAI) rise to 0/9 and GC content increase to 54.9. The optimal vaccine combination for the chimeric protein was determined to be CTxB-BLF1-StxB. Expression of recombinant protein in E. coli led to the production of a chimeric protein with 48 kDa molecular weight.

Conclusion

The findings of the current study revealed that this antigen can be raised as a recombinant vaccine candidate which can be due to such factors as a property of the ability to stop protein synthesis in BLF1, CTxB adjuvant, and delivery of StxB protein.
Keywords
Burkholderia pseudomallei
BLF1 toxin
Melioidosis
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