This study was performed at Department of Biochemistry, Institute of Biochemistry, Biotechnology and Bioinformatics, The Islamia University of Bahawalpur, Pakistan.

Selected genes, vector and bacterial strains

All the selected genes were obtained in cloned pET vector form through BeiResources, National Institute of Allergy and Infectious Diseases (NIAID), USA. pVAX11™ (Catalog # V260-20) used as mammalian expression vector and the bacterial strain DH5α used for transformation was purchased from Invitrogen, Thermo Fisher Scientific™, USA.

Primer Designing

Primers were designed by adding appropriate restriction sites in both forward and reverse primers by flanking the start and stop codons. Kozak consensus sequence was also inserted in forward primers of each gene prior to Restriction site for appropriate initiation of translation. The complete sequence along with restriction sites of all primers are given in Table # 1.

Expression cloning of the selected genes

All the selected genes were amplified through Polymerase Chain Reaction by using DreamTaqᵀᴹGreen PCR–Master Mix (Catalog # K1081) from Thermo Scientific. The PCR profile comprised an initial denaturation step at 95°C for 5 minutes, succeeded by 35 cycles of denaturation at 95°C for 45 seconds, annealing at a specific Tm (as indicated in the Table # 1) for 1 minute, and extension at 72°C for 1 minute. A final extension was conducted at 72°C for 10 minutes. PCR reaction of Control DNA (700 bp) with its Control Primers was also run parallel to all gene reactions as positive control. All amplified genes and pVAX1 vector were double digested with their specific restricted endonucleases enzymes and purified. Double digested pVAX1 were also treated with rSAP enzyme to prevent it from self-ligation. After confirmation and quantification of double digested genes and vector, both were ligated to each other using T4 DNA ligase enzyme.

Once the accomplishment of ligation was confirmed through electrophoresis, the ligated product was immediately transformed into competent cells which were prepared by TSS method, following the protocols given by Chung et al [20]. The transformed ligated products were spread over kanamycin positive respective labelled plates one by one. Positive and negative control plates were also structured and incubated at 37˚C for overnight. Next morning the plates were observed, and transformation was confirmed through appearance of colonies on plates.

A single colony from the center of each plate was picked and added in kanamycin positive autoclaved LB media and placed in shaking incubator for overnight. Next day the growth was observed, and plasmid extraction was performed using Thermo Scientific™ GeneJET Plasmid Miniprep Kit catalog#K0502. The extracted plasmid was confirmed by agarose gel electrophoresis.

Clone Confirmation though PCR And Restriction Digestion

Colony PCR as well PCR of extracted plasmid was done using both gene and pVAX1 specific primers for confirmation of constructs. Further, these constructs were also confirmed through single and double digestion with different restriction endonuclease enzymes.

Sequencing of all positive Constructs

Final confirmation of the positive clones was done, by sending purified plasmid constructs to BGI Tech Solutions Hongkong for DNA sequencing. After sequence analysis, clones which have the right sequence were selected and their glycerol stocks were made.

All genes PCR amplification

All the selected Mycobacterium specific genes were amplified by PCR using sequence specific complementary reverse and forward primers. The PCR conditions and annealing temperature of primers were optimized. The result of PCR amplification of all the selected genes are shown in Figure 1.     

Restriction Digestion

The amplified genes and extracted pVAX1vector were double digested with specific restriction digestion enzymes which are mentioned in table no. 1. pVAX1was also restricted with each single enzyme parallelly to check the enzyme activity as positive control, shown in Figure # 2, 3 & 4.

Ligation and transformation

After purification and quantification of double restriction digestion of gene and vector, both were ligated to each other using T4 DNA ligase enzyme. Quantification was done by using EPOCH microplate spectrophotometer through nanodrop. The ligated products were immediately transformed into competent cells and spread over kanamycin positive agar plates overnight. Self-ligated pVAX1 plasmid was transformed as positive control while linear single cut pVAX1 plasmid was transformed as negative control, shown in Figure # 5.

Confirmation of Constructs though Restriction Digestion and PCR

The extracted constructs were confirmed through PCR using both gene and pVAX1 specific primers, shown in Figure # 6. Single and double digestion with different restricted enzymes was performed to further confirm the constructs and successful transformation of desired gene into the vector, shown in Figure # 7-10.

Sequence Analysis of Constructs

Proper orientation of the specific gene into the vector was finally verified through sequence analysis performed by BGI Tech Solutions Hongkong.

Figures & Tables

Discussion

Reduction of tuberculosis incidence and development of modern and more effective drugs as well as vaccine against tuberculosis is the main target of WHO tuberculosis eradication program [21]. As the whole genome sequencing of M. tb is completed, more than hundred DNA vaccines have been designed and studied in various animal models but the protective antigen for TB is still not clear [22]. This deficit has created the limitation to the development of tuberculosis vaccines [23].

Formerly, various M. tb secreted proteins were regarded as suitable candidate vaccines but not all were effective. Some DNA vaccine encoding genes such as Mpt32, Bfrb, esat6 were produced and have been reported to stimulate the antibody response [24,25].

Another study including the development of subunit-based DNA vaccines was performed by using various genes of TB like cfp10, hspx, ag85a, ag85b and ag85c, which reports very strong immune response in mouse model in alone or combination with BCG [26]. Therefore, many gene encoding vaccine have shown very promising level of protection in different animal models and can be suitable candidate for TB vaccine.

In the present study, five genes related to mycobacterium were used for the designing and production of DNA vaccine including Rv1908c/KatG, Rv0440/GroEL2, Rv3418c/GroES, Rv0350/DnaK and Rv0934/PstS. All these genes were reported to have important biological functions and are structural parts of the cell, suggesting being very significant for survival of bacterium and also for taken to be as antigen [27-31]. In recent studies Rv1908c/KatG was selected and used by different groups and proved the importance of this gene in bacterial pathogenesis and DNA vaccine. Mycobacterial enoyl reductase inhibition as clinically evidenced by isoniazid, the most potent TB drug, KatG provides the most effective methods for eliminating M. tb [32]. In another study recombinant DnaK (Rv0350) and MPT83(Rv2873) gene constructs was tested on TB patients and found efficient in provoking the immune response against M. tb. Through genetic recombination and protein purification technology, these two antigenic proteins were cloned and purified. Blood samples were obtained from TB patients, non-TB patients with other pulmonary diseases and healthy subjects. In the immunological properties of stimulating humoral and cellular immunity, the results revealed that the sensitivity and specificity of both MPT83 and DnaK proteins were 76. 30％ & 58. 95％ and 77. 80％ & 66. 67％ respectively [33]. Another study presents cloning the genes of the immunodominant antigen of M. tb i.e. PstS and its T cell epitopes (PstS1p). This study revealed the immunity and immunogenicity of both PstS1 and its epitope PstS1p, which significantly produced higher antigen-specific IgG antibodies in mice serum, indicating increased antibody response. They also promoted proliferation of CD4+ T cell by stimulating the T helper 1 response, indicating the cruciality of this antigen as promising DNA vaccine candidate for controlling tuberculosis [34].

This study was victorious in cloning of five selected genes of Mycobacterium into mammalian expression vector pVAX1and making the very desirable candidate as DNA vaccines. These gene encoding constructs may be used in various animal models to verify the immunization and also for the challenge studies against TB in future experiments.

Conflict of Interest

The authors have no conflicts of interest to declare. 

Author Contributions

Muhammad Mohsin Zaman: Conceived idea, conducted the research, collected, analyzed and interpreted the data and wrote the article.

Mirza Imran Shahzad: Planned and supervised the research, edited and approved the final manuscript

Aeman Jilani: Helped in conducting research and writing of article, collected, analyzed and interpreted the data.

Areeba Yousaf: Helped in conducting research and writing of the article.

Ansah Bashir: Helped in conducting research and writing of article

Saher Riaz: Helped in conducting research and writing of article

Gildardo Rivera Sanchez: Financial support the research, edited and approved the final manuscript.

References