Construction of an Expression Vector Containing Mtb72F of Mycobacterium tuberculosis

Abstract

Objective: Despite using the Bacille Calmette Guerin (BCG) vaccine, tuberculosis (TB) is still a worldwide disease that kills 2-3 million people each year. Developing a new and more effective vaccine is one way to possibly reduce the morbidity and mortality of TB. The Mtb72F vaccine is one of the important subunit vaccines applied in human clinical trials. In this study, we have constructed an expression vector that contains the Mtb72F fragment with some new modifications.

Materials and methods: In this experimental study, Mtb32N and Mtb39 fragments were amplified by polymerase chain reaction (PCR) using specific primers and inserted into pET21b\Mtb32C. Colony-PCR, restriction enzyme analysis, and DNA sequencing were used to confirm the accuracy of the cloning. We used Western blot to verify the desired protein expression.

Results: The amplified fragments showed the desired size in PCR and digestion methods, and protein expression was confirmed using a monoclonal antibody.

Conclusion: Our modification made it possible to insert another gene or gene fragments into the Mtb72F vector for developing new constructs. In addition, our data has shown that the placement of the histidine tag in the carboxyl- (C-) or amino- (N-) terminal part of a protein may influence protein expression and/or stability.

Keywords: Mtb72F; Mycobacterium tuberculosis; Vaccine.

Fig 1

PCR products of the amplified fragments. Mtb32N fragment 632bp (A), Mtb39 fragment 1197bp (B), and 100 bp DNA size marker (M).

Fig 2

Restriction enzyme analysis of the final construct using different enzymes: Lane A (arrow): double digestion using NdeI and EcoRI enzymes (1600 bp fragment consisted of Mtb32C and Mtb39); Lane B (arrow): double digestion using BamHI and EcoRI enzymes (1200 bp fragment of Mtb39); Lane C (arrow): double digestion using NdeI and HindIII enzymes (2200 bp fragment consisted of Mtb32C, Mtb39, and Mtb32N).

Fig 3

Western blot results to confirm protein expression. Lanes 1 and 2: E. coli BL21 containing an empty pET21 vector. Lanes 3 and 4: E. coli BL21 consisting of the Mtb72F protein.