Designing, cloning and expression of ctxB– tcpA-c-cpe gene in E.coli as a cholera vaccine candidate

Subject Areas : Microbial Biotechnology

Negar Souod ¹ , Mohammad Kargar ² , Mohammad Hossein Hoseiny ³ , Mojtaba jafarinia ⁴

1 - ....
2 - Department of Microbiology, Jahrom Branch, Islamic Azad University, Jahrom, Iran
3 - Immunology Department, Razi Vaccine and Serum Research Institute, Shiraz Branch, Agricultural Research, Education and Extension Organization (AREEO), Shiraz, Iran.
4 - 1Department of biology, Marvdasht branch, Islamic Azad University, Marvdasht, Iran

Received: 2020-05-16 Accepted : 2020-10-16 Published : 2021-05-22

Keywords: Vibrio cholorea vaccine, C terminal of Clostridium perferingenes toxin, tcpA gene, ctxB gene,

Abstract :

Background & Objectives: TcpA protein and B subunit of cholera toxin are the most important virulence factors of Vibrio cholorea. In the current survey, we applied the C-terminal of          clostridium perferingenes toxin as a delivery system to bind the CtxB-TcpA fusion as an antigen,      cloning it in a prokaryote vector, evaluated the level of  expression.Materials & Methods: In experimental survey, the ability of a constructs based on                    CtxB-TcpA-C-CPE with complete protein sequences of each protein was studied. After             amplification of tcpA, ctxB, and c-cpe genes using PCR, they were cloned in expression plasmids. For fusion protein, all of the three protein sequences were constructed with linker. After             expression, the proteins were purified and then confirmed using immunoblot methods.  Results: This fusion protein consists of 484 amino acids. PCR amplification for the c-cpe, tcpA and ctxB genes amplified 381, 375 and 675 bp; respectively. The result of enzymatic restrictions and sequencing indicated the exact homology of the synthesized proteins and the others submitted in NCBI. According to the SDS-PAGE results, the TcpA, CtxB, and C-CPE proteins were 15, 25 and 25 kD respectively.Conclusion: According to physicochemical results, this fusion protein may be suitable candidate as a vaccine, however; further experimental trials are needed to approve this conclusion.