1. Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is responsible for 9 million new cases of active TB and 1–1.5 million deaths annually ^[1]. It is estimated that nearly 2 billion people suffer from latent TB around the world, which potentially become an active TB. The only vaccine currently used is BCG, which has been developed since almost a century ago. BCG is given in newbornes routinely in TB endemic countries. However, the effectiveness of BCG was reduced in adolescents, and the vaccine does not consistently prevent the development of active pulmonary TB in adults (efficacy estimated between 0 and 80% with the lowest efficacy rates often found in countries with the highest burden of TB) ^{[2, 3, 4]}. Thus, there is an urgent need for a new TB vaccine to either boost immunity primed by BCG or replace BCG ^{[5, 6]}.

The variation in sequence and length of C-terminal region among members of the unique PE (Pro-Glu) and (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation. Based on in-silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, which is indicating the expression of this ORF (Rv2430c) at the mRNA level. Our results reveal that this ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or , pointing to the immunodominant nature of the protein ^[7].

Cloning of ORF Rv 2430 performed on pGEM-T cloning vector and host Escherichia. coli JM 109. It is expected that this gene can be used as candidate antigens for TB vaccine search.

2. Materials and Methods

The cloning vectors pGEM-T Easy (Promega) was used. The strains M. tuberculosis was obtained from clinical isolate from Indonesia. E. coli JM 109 was obtained from Center For Biomedical and Basic Technology of Health.

The E. coli JM 109 were incubate with stirring over night in LB medium in the presence of ampicillin (1μg/ml) at 37°C. E.coli JM109 competent cells prepared in advance by the CaCl2 method ^[8] modified.

The following primer was design by ^[7]

Rev 5’-AAGCTTCTAAGTGTCTGTACGCGATGA-3’

Fwd 5’-GGATCCATGCATTTCGAAGCGTAC-3’

The reactions were performed with 1 μl of template in a total volume of 25 μl. Generally, the profiles consisted of a hot start at 94°C, an initial denaturation at 94°C for 1 minute, a denaturation at 94°C for 30 second, an annealing at 55°C for 1 minute and an extension step at 72°C for 40 second. After 25 cycles an extension was carried out at 72°C for 7 minutes. products were examined by agarose gel electrophoresis and staining with ethidium bromide.

Isolation of DNA and transformation of the E. coli JM 109 cells were performed as described in the guidebook ^[8], with the following modifications. Transformed cells were spread on the appropriate indicator plates containing ampicillin. Colonies were scored for phenotype on Luria Bertani agar plates after 24 hours at 37°C.

Fragment of the ORF Rv 2430c were obtained by using two primers pair and DNA of the M. tuberculosis Indonesian strain as a template. The pGEM-T vector and PCR product were cut with BamHI and HindIII, mixed, and treated with T4 DNA ligase. The resulting recombinant plasmid pGEM-T-Rv2430c was transformed in E. coli JM 109.

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Figure 1. Ligation scheme of ORF Rv2430c into cloning vector pGEM T-Easy

3. Results and Discussion

The results of amplification of ORF Rv 2430 observed by electrophoresis agarosa. A band was revealed which corresponded to the gene with the apparent of 597 bp. It is appropriate that obtained Choudhary et al, 2003 that 597bp band was observed upon staining with ethidium bromide, indicating that the ORF was expressed at the mRNA level in the liquid cultures of M. tuberculosis

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Figure 2. Purification of product: 1) Marker 100 bp, 2) ORF Rv 2430c

Isolation of plasmid were performed according to the procedure instructions (BioRad). Characterization was done by migration analysis, restriction analysis, and PCR analysis. Migration analysis was done by comparing pGEM-T (3000bp) and pGEM-T-Rv 2430c (3597bp). A band shows difference length while pGEM-T-Rv2430c had slower migration than pGEM-T (Figure 3).

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Figure 3. Migration analysis: 1) pGEM-T: 3000 bp, 2) pGEM-T-Rv2830: 3597bp

This suggests that the plasmid without insert (pGEM-T) will move faster than the recombinant plasmid (pGEM-T-Rv 2430). This means that the inserts ORF Rv 2430 has been successfully ligation into the vector pGEM-T.

Restriction analysis was done using 2 restriction enzymes BamHI and HindIII in pGEM-T-Rv2430c. By electrophoresis, a linier band was showed a different shape with uncut recombinant plasmid which has supercoiled and relaxed band (Figure 4).

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Figure 4. Restriction analysis: 1) pGEM-T-Rv2430c (3597bp) uncut; 2) pGEM-T-Rv2430c (3597bp) cut; 3) Marker 1 kb

Uncut plasmids can be in two forms: relaxed and supercoiled (or superhelical). The lane with the digested plasmid has a single band, while the lane with the undigested sample has three bands. If the lower band had moved most rapidly because it was a smaller fragment than the others, then we would expect that it would be much dimmer than the others because, in general, less fluorescent dye is intercolated by smaller fragments. However, in this case the lower band is much brighter than the other bands, either because the supercoiled shape holds dye better or because a higher fraction of the molecules in that lane were in the supercoiled form than in other forms ^[9].

analysis was done by using exactly the same cycle as it mentioned before for amplified the Rv 2430c gene. Electrophoresis showed that plasmid recombinant pGEM-T-Rv2430c contain the Rv 2430c gene as DNA insert was 597 bp (Figure 5).

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Figure 5. analysis: 1) Rv 2430c :597bp 2) Marker 100 bp

4. Conclusion

Cloning ORF of Rv 2430c M. tuberculosis Indonesia isolates to Escherichia coli JM 109 has been successfully and characterized insert by , migration analysis and restriction analysis.

Acknowledgments

The authors would like to thank Kemenristek Dikti and Center for Biomedical and Basic Technology of Health, which has funded this research through a Consortium of TB Vaccine.

References

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