CHARACTERIZATION OF RECOMBINANT PROTEIN ESAT-6 Mycobacterium tuberculosis AS IMMUNODIAGNOSTIC LATENT TUBERCULOSIS

Rosana Agus1and Debbie S.Retnoningrum2 Faculty of Science, Universitas Hasanuddin1

School of Pharmacy, Institut Teknologi Bandung2

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2. Laal, S. dan Y.A.W. Skeiky. 2005. Immune-based methods. In Cole ST, Eisenach KD, McMurray.

3. Xu, J., W. Xu, X. Chen, D. Zhao, Y. Wang. 2008. Recombinant DNA vaccine of the early secreted antigen esat-6 Mycobacterium tuberculosis and Flt3 ligand enhanced the cell mediated immunity in mice. Science Direct 26(35) : 4519-4525.

4. Nagel, M.,2006, Purification of Polyhistidine-Tagged Proteins User Manual, Germany and International Macheret-Nagel Gmbh & Co.KG

Purification protein

Clone pET32b-ESAT-6 in E.coli BL 21

Culture in medium LB +Amp+ IPTG

Overproduction protein

Reactivity test protein with

serum

Result

Abstract

BCG vaccine has been used since 1921 to prevent tuberculosis. However, the effectiveness of BCG began to doubt because of continuing high tuberculosis sufferers in the world, including Indonesia. Although nearly all of Indonesia population had been vaccinated with BCG, but tuberculosis remains increased and made Indonesia ranks fourth in the world after India, China, and South Africa. Therefore, the necessary replacement BCG vaccine is effective that had been done to eliminate tuberculosis. One of the antigens are widely studied as a tuberculosis vaccine candidates is early secreted antigenic target (ESAT-6). This protein is secreted by Mycobacterium tuberculosis in the early phase of growth, and has been shown to induce the production of interferon gamma by CD8 T cells. The purpose of this study was to characterized the recombinant protein ESAT-6 of M. tuberculosis as a vaccine antigen for TB. The research method consisted of over-production of proteins, protein purification and immunoreactivity test with serum of patients tuberculosis. Results were obtained ESAT-6 recombinant protein recognized by serum of patients with TB, but not recognized by the serum of healthy people. Keywords: ESAT-6, vaccination, tuberculosis, protein

Tuberculosis (TB) remains a major global health problem. Human TB is the most frequent cause of death from a single infectious agent, being responsible for about two million people die every year worldwide [1]. About 90% of people who get infected with TB develop a latent TB infection. People who have latent infections do not have TB symptoms and cannot spread the infection to others, but they are at risk of developing an active infection that is both symptomatic and contagious. Latent TB infection was difficult to detect due to the non specific symptom. TST (Tuberculin Skin Test) was the only test used to detect latent TB infection this

past 100 years [2] but found to have low spesificity. Furthermore, immunodiagnostic complement test was needed by using host cellular response and antigen identification. The mycobacterial protein ESAT-6 (molecular weight 6 kD), which is also secreted during the early stage of the mycobacterial growth, was encoded by Rv3875 (288bp) and found as immunodominant antigen because it contains epitops recognizable by protective T-cells with high specificity and sensitivity in TB patient serum [3]. ESAT-6 increase IFN- which can be measured by detection kit. Despite the potential of ESAT-6 in detecting latent TB infection, ESAT-6 resulted from Indonesian isolate has not been produced. In order to express and produce ESAT-6, from Indonesia isolate. Our previous studies have successfully ligating ORF ESAT-6 into pET32b

expression vector and transformation into host cell Escherichia coli BL 21 (DE3). Stages of research is important so that ORF ESAT-6 can be expressed into protein. In this study will be conducted over expression and purification of recombinant protein pET-32b-ESAT-6 and was tested with tuberculosis serum.

Figure 1. Overproduction of protein His(6)-ESAT-6 Note: 1 = marker protein ESAT-6 2 = non-induced (NI) 3 = ESAT-6 induction (I) 4 = pET-32b (NI) 5 = pET-32b (I)

Figure 2. Purification of protein His(6) -ESAT-6 Note: 1 = marker protein 2 = flow through (ft) ESAT-6 3 = W1 (wash) ESAT-6 4 = W1 pET 5 = E1 (elution) ESAT-6 6 = E2 ESAT-6 7 = E3 ESAT-6 8 = ft pET 9 = E2pET 10= E3 pET

Figure 3. Test reactivity protein His(6)-ESAT-6 with normal serum Note : K = negative control W1 = wash protein 1 E2 = elution protein 2

K

W1

E2 K

E2

Figure 4. Test reactivity protein His(6) -ESAT-6 with serum patient TB Note: K = negative control W1 = wash protein 1 E2 = elution protein 2

Overproduction of protein with 0.5 mM IPTG obtained recombinant fusion protein His (6)-ESAT-6 is 24 kDa. Test reactions ESAT-6 protein in serum of patients with active tuberculosis and serum of healthy people with Dot blot showed that serum TB dots appeared clearer and brighter than the serum of healthy people.

The purpose of this study was to characterize the recombinant protein ESAT-6 as antigen for immunodiagnostic latent tuberculosis.

The authors would thanks to Kementerian Riset dan Teknologi which was fund this research .

Conclusion