Designing and Construction of a Cloning Vector Encoding mtb32C and mpt51 Fragments of Mycobacterium tuberculosis as a DNA Vaccine Candidate.

BACKGROUND &
OBJECTIVE: Tuberculosis (TB) remains a major cause of death around the world. Bacillus Calmette Guérin (BCG) is the only vaccine used in TB prevention that has a protective effect in children, but its effectiveness declines in adults. Design and development of new vaccines is the most effective way against TB.The aim of this study was to design and construct a DNA vaccine encoding mtb32C and mpt51 fusion genes of Mycobacterium tuberculosis.
METHODS: First, mpt51 fragment was amplified by PCR method. The pcDNA3.1+/mtb32C plasmid was transformed into E. coli JM109 and then extracted. The mpt51 gene and pcDNA3.1+/mtb32C plasmid were both digested with EcoRI and BamHI restriction enzymes followed by ligation of mpt51 fragment into the digested vector. The recombinant plasmid containing mtb32C and mpt51 was subsequently transformed into competent E. coli TOP10 strain. The clones were confirmed by colony-PCR, restriction enzyme digestion and sequencing.
RESULTS: Using agarose gel electrophoresis, a 926 bp fragment corresponded to mpt51 was observed. Digestion of the vector pcDNa3.1+/mtb32C and mpt51 gene was confirmed by electrophoresis. Then, the pcDNA3.1+/mtb32C plasmid was extracted. Sequencing results confirmed the accuracy of the desired plasmid.
CONCLUSION: In this study, we constructed a cloning vector encoding mtb32C/mpt51 gene of M. tuberculosis. The eukaryotic expression of this vector can be confirmed in future studies. It can be considered as a DNA vaccine in animal models later. Successful cloning provides a basis for the development of new DNA vaccines against TB.

Introduction

Tuberculosis (TB) is a major and contagious infectious disease with over eight million new cases per year (1, 2). According to the World Health Organization (WHO), one-third of the world’s population is estimated to be infected with Mycobacterium tuberculosis (M. tuberculosis). Of these, 5-10% of the latent population will develop clinical manifestations of TB, of which 2-3 million people die, annually (3, 4). Bacillus Calmette Guérin (BCG) vaccine which has been administered to more than one billion people worldwide, has shown its significant effectiveness against tuberculosis meningitis and miliary tuberculosis in youngsters but not in adults (5). On the other hand, in the 1980s, it became clear that those infected with AIDS are susceptible to M. tuberculosis infection. These reasons and the higher risk of disseminated BCG tuberculosis in HIV-infected neonates encouraged scientists to develop new vaccines against tuberculosis. These vaccines should stimulate Th1 cells to increase the capacity of macrophages against the disease (6).

MTB32A is a serine protease extracted from culture filtrate proteins (CFP) of M. tuberculosis. This protein plays a major role in the bacterial survival and pathogenesis. The C-terminal of MTB32A is capable of eliciting TCD8+ and resistance to TB (7).

Mpt51 is a major secreted protein of the M. tuberculosis, which is found in the genomes of M. tuberculosis, M. leprae, M. avium, and M. bovis BCG. This protein attaches to the fibronectin of the extracellular matrix of the host cells and might have a major role in the virulence of the bacterium (8).

Mpt51 is closely related to the Ag85 complex of M. tuberculosis and has a high tendency towards fibronectin. It has mycolyl transferase activity that is necessary to make trehalosedimycolate (cord factor), and a structure necessary to maintain the integrity of the cell wall (9). The evidences suggest that mpt51 plays an important role in adhering to host tissue through binding to fibronectin (10). The aim of this study was to design and construct a DNA vaccine encoding mtb32C and mpt51 fusion genes of M. tuberculosis.

Materials and Methods

Bacterial strains and DNA extraction

Standard strain of M. tuberculosis H37Rv (ATCC 27294) was purchased from Tuberculosis Reference Laboratory of Shariati Hospital (North-East of Mashhad) and cultured on Löwen stein–Jensen medium at 37ºC for a period of four weeks. Then, DNA was extracted by boiling procedure; in brief, 4-5 colonies of M .tuberculosis H37Rv were suspended and homogenized in 500 μl sterile PBS buffer. Suspended bacteria were boiled for 15 min. The supernatant was used as DNA template in the PCR reaction (11).

Primer designing and PCR method

DNA encoding mpt51 sequence is located in RD1 region and called mpt51, mpb51, fbpC1or Rv3803c. To develop specific primers for the amplification of mpt51, the DNA fragment encoding mpt51 gene consisting of 926 nucleotides was retrieved from NCBI GenBank. Based on this sequence, the forward and reverse primers; (5’ -AGATAAGGATCCACCATGGGAAAGGGTCGGTCGGCGCT-3’) (5’TCGGCAGAATTCTTAGCGGATCGCACCGACGATATCGC-3’) were designed using Gene Runner software. The PCR mixture contained 10X PCR buffer, 0.1mM dNTP, 1.5 mM MgCl2, 1 μl DNA (100ng/μl), 10 pmol of each primer, 1.5 unit of Taq DNA polymerase (Fermentas, UK). The adequate amount of double distilled water (DDW) was added to the reaction. The negative control contained all the PCR reagents without the template DNA. Amplification was done on an Eppendorf thermocycler (UK) using the following cycling conditions: 95°C for 30 sec, 35 cycles at 57°C for 45 sec, 72°C for 30 sec followed by a final extension at 72°C for 3 min. The PCR product electrophoresis was performed on low melting 1% agarose gel (Fermentas, UK). The desired band was purified by DNA extraction kit according to the manufacturer’s protocol (Bioneer, Korea).

Construction of recombinant plasmid pcDNA3.1+/mtb32C/mpt51: Digestion, ligation and transformation

PcDNA3.1+ vector containing mtb32C (pcDNA3.1+/mtb32C) was previously constructed in our laboratory (12). The vector was transformed into E. coli JM109 and then purified using plasmid extraction kit (Bioneer, Korea).

Both purified PCR product and pcDNA3.1+/mtb32C vector were digested by EcoRI and BamHI restriction enzymes (Fermentas, UK). The reaction mixture contained 20 μl of mpt51 DNA (25 ng/μl), 5 μl H Buffer, 2 μl EcoRI (10 u/μl), 2 μl BamHI (10 u/μl) and 31 μl nuclease-free DDW. The vector concentration for enzyme digestion was 100 ng/μl. The digested products were run on the gel and purified by gel extraction kit (Bioneer, Korea). In order to insert the mpt51 gene into pcDNA3.1+/mtb32C vector, ligation was performed with T4 DNA ligase (Fermentas, UK). The ligation reaction included: 6 μl (100 ng/μl) of pcDNA3.1+/mtb32C, 12 μl (100 ng/μl) of mpt51, 2.5 μl of T4 DNA ligase buffer, 2 μl of 5u/μl T4 DNA ligase, 2 μl of polyethylene glycol (PEG 4000) and 0.5 μl of DDW. The competent E. coli TOP10 strain was prepared using cold solution of CaCl2/MgCl2 (0.1405.Construction of Vector Encoding mtb32C and mpt51 M) as described previously (12, 13). Transformation was performed using heat shock method and the transformed bacteria were cultured on LB agar containing 100 µg/ml ampicillin (14).

Confirming the desired construct

The colonies grown on the LB agar plates containing ampicillin were used as DNA template in PCR reaction using mpt51 gene primers with the same conditions that were optimized for mpt51 gene. The accuracy of designed pcDNA3.1+/mtb32C/mpt51 recombinant vector was confirmed by sequencing.

Results

The amplification of mpt51 gene was performed by the PCR method using specific primers and a fragment with 926 bp in length was observed on 1% w/v agarose gel electrophoresis (Figure1).

Figure 1

Agarose gel electrophoresis of PCR product showing the amplification of mpt51 gene. Lane 1: band of 926 bp corresponds to mpt51gene; Lane C-: negative control; Lane M: 100 bp DNA size marker (Fermentas, UK)

The PCR product band related to mpt51 gene was purified (Figure2). Recombinant vector pcDNA3.1+/ mtb32C and mpt51 gene were digested with EcoRI and BamHI restriction enzymes, then ligation was performed using T4 DNA ligase enzyme. The ligation product was transformed into competent E. coli TOP10 strain (7, 15). The colony PCR was performed to select recombinant vectors containing mpt51 gene (Figure 3). The sequencing results showed 100% sequence homology without any gap and mutation in our final recombinant plasmid compared to recorded sequences of H37Rv mtb32C and mpt51 fragments.

Figure 2

Agarose gel electrophoresis of purified PCR product of 926 bp mpt51 using DNA extraction kit from gel, Lane M: 100bp DNA size marker (Fermentas, UK); lane 1: purified mpt51gene; Lane C-: negative control

Figure 3

Agarose gel electrophoresis of colony-PCR showing recombinant vector pcDNA3.1+/mtb32C/ mpt51 using mpt51-specific primers. Lane C-: negative control; Lanes 1 and 2: positive colonies; Lane M: 100 bp DNA size marker (Fermentas, UK)

Discussion

The previous attempts using alternative antigens of mycobacteria other than BCG have not been acceptable. The availability of cloned genes and suitable vectors has paved a new way to synthesize mycobacterial protein antigens individually by transfection to mammalian cells (16).

Studies have shown that, DNA vaccination of heavily infected mice with TB can alter the inefficient immune response to an effective one that destroys bacteria. Immunotherapy with DNA vaccine in combination with conventional antibacterial therapy might result in more efficient treatment of infection in humans. The main effects of this vaccination were stimulation of CD4+ T cells and production of IFN-γ together. As a result, those antigens which are more capable of stimulating T cell responses against M. tuberculosis would be more immunogenic, hence, a better candidate for vaccine development. The earlier studies have shown that recombinant fusion protein mtb32/mtb39 vaccine from M. tuberculosis provide better immune responses compared to each of the proteins alone. Thus, as a candidate vaccine, the cloning of various antigens with mtb32 may improve the host immune responses compared to any single antigen (7).

For this purpose, designing and construction of two other fusion DNA vaccines: one encoding tb10.4 and mtb32C-hbha and another encoding ag85A and tb10.4 fusion of M. tuberculosis were conducted in Antimicrobial Resistance Research Center at the University of Mashhad (7, 17).

Conclusion

In this study, mtb32C/mpt51 recombinant vector was constructed successfully. It should be noted that the termination codon of mtb32C has been removed. The gene encoding mpt51 was added in frame to mtb32C sequence. More broadly, research is needed to determine immune responses to this vaccine in animal models.