Makara J. Health Res.

, 2018, 22(1): 8-13

doi: 10.7454/msk.v22i1.7954

Development of a Tuberculosis Vaccine Seed: Construction of Resuscitation-

Promoting Factor B DNA Vaccine and its Expression in Vitro and in Vivo

Ratih D Saraswati1, Andriansjah Rukmana2*, Fithriyah2, Aprilia Rakhmawati1

1. Biomedical Science Programme, Faculty of Medicine, Universitas Indonesia, Jakarta 10430, Indonesia
2. Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta 10320, Indonesia
*
E-mail: [email protected]

Abstract
Background: Tuberculosis (TB) is a chronic infection disease caused by Mycobacterium tuberculosis (Mtb) and has a
high death-rate worldwide. Bacillus Calmette-Guerin is the only TB vaccine which is currently available with several
drawbacks, such as its different efficacy for different individuals, lack of protection for lung TB in adults and
subsequent reactivation which lead the research for novel TB vaccine approach. Resuscitation-promoting factor (rpf)
protein in Mtb is a protein cluster which play a big role in TB dormancy during latent infection. Member from this
cluster protein is rpfB which shows the greatest biological and immunological characteristics among other proteins in
the rpf family, now is widely explored as novel TB vaccine candidate. Methods: In this study, the rpfB gene of the Mtb
Beijing strain was amplified using PCR and then cloned into pcDNA3.1 plasmids. The ability of recombinant pcDNA-
rpfB to induce humoral immune response was tested through Balb/C mice immunization. Results: A positive
recombinant rpfB protein ~66 kDa was detected through western blot analysis using immunized mice sera. Meanwhile,
recombinant pcDNA-rpfB was transfected in to CHO-K1 mammalian cell line and recombinant rpfB antigen expression
was confirmed through immunostaining. Conclusions: Therefore, we have succesfully express the recombinant rpfB
proten of M.tb strain Beijing in mammalian expression system which proven to be antigenically induced humoral
immune response in mice model.

Keywords: CHO-K1, DNA vaccine, Mycobacterium tuberculosis, rpfB gene

Introduction England to 0% in Chinglepur, India and some places in

the USA.5,6
Tuberculosis (TB) is a chronic infectious disease caused
by Mycobacterium tuberculosis. According to the 2015 The BCG vaccine is useful to protect from severe
World Health Organisation (WHO) data, TB is one of childhood TB, but the immune effect it produces only
the top 10 infectious diseases worldwide, with a death- lasts for 10 to 15 years and does not provide protection
rate of 1.8 million people yearly, higher than that of against lung disease in adults.4 In addition, the BCG
HIV and malaria. Countries with the greatest number of also does not provide protection against TB latent
TB patients include India, Indonesia, China, Nigeria, infection and its subsequent reactivation.5 This has
Pakistan and South Africa.1 Most individuals infected prompted new research to develop anti-TB vaccines that
with M. tuberculosis experience latent TB infection, are more effective.
meaning they do not show symptoms and do not
transmit the disease. In latent TB patients, bacteria are Preliminary studies and bioinformatic analyses have
dormant and do not cause clinical symptoms but can be identified some M. tuberculosis genes as possible new
reactivated.2 vaccine candidates. Possible vaccine candidate antigens
may originate from the resuscitation-promoting factor
TB can be prevented by vaccinating those who have not (rpf) gene family. The rpf genes expresses rpf proteins
been infected by the M. tuberculosis bacteria. The that play a role in bacteria recovery (resuscitation) in the
Bacillus Calmette–Guerin (BCG) is the only anti-TB latent to replication phases.7 M. tuberculosis expresses
vaccine used worldwide nowadays. This vaccine five different Rpf proteins, namely RpfA–E, all of
originated from an attenuated Mycobacterium bovis which have resuscitation activity and are immunogenic
strain3. BCG is a very secure vaccine, relatively cost- in mice.7 RpfB protein, one of the five Rpf proteins
efficient and stable at room temperature,4 however, its produced by M. tuberculosis, plays an important role in
protection capability varies, ranging from 77% in dormant bacteria recovery and growth. Analysis of

8 April 2018 | Vol. 22 | No. 1

 Development of a Tuberculosis Vaccine Seed 9

RpfB protein composition has shown that this protein is Mouse immunisation. Preparation of plasmid for
a membrane protein with B- and T-cell multiple immunization were done by growing E.coli DH5
epithopes.8,9 Previous research showed that the RpfB strain consist of recombinant plasmid or pCDNA3.1
protein has the highest biological and immunological itself in 100 mL LB medium with 100 g/mL ampicillin
characteristics among other the Rpf family proteins.10 at 37 oC overnight. The bacterial cells were harvested
Deletion of the M. tuberculosis rpfB gene causes a delay and then plasmid were isolated using a GeneJET
in reactivation in mice infected with M. tuberculosis. Plasmid Midiprep Kit (Thermoscientific). Concentration
Furthermore, mutations in this gene can reduce the of plasmid were adjust to 100 µg in 100 µL of TE
growth levels of the M. tuberculsis H37Rv strain. This buffer. Immunisation was performed on 6 to 8-week-old
makes RpfB a suitable antigen for the development of a male Balb/C mice. The mice were divided into three
new or as complementary TB vaccine.10 treatment groups containing six mice each. Mice were
injected with pcDNA3.1 plasmid only, Tris-EDTA (TE)
Meanwhile, the newest method in vaccine development or the pcDNA3.1-rpfB recombinant plasmid. Plasmids
uses DNA as a vaccination platform, known as a DNA were injected intramuscularly using a disposable syringe
vaccine. DNA vaccines consist of a bacterial plasmid with a 27 G needle on either the right or the left thigh
with a strong eukaryotic promoter gene (such as an muscle. A booster was performed on days 7, 14 and 21
enhancer element from cytomegalovirus), a gene that and blood was also drawn from the mice using the retro-
encodes an immunogenic protein, a polyadenylation orbital technique. Mice were anaesthetised via eter
signal and a transcriptional termination sequence.11 inhalation before blood samples were taken. The blood
Although there have been many studies of the RpfB sample volume was 130 µL, around 10% of the total
protein, to our knowledge, there has been no research blood volume. Fifteen days after the last immunisation,
into the development of a TB vaccine based on DNA blood was redrawn using the retro-orbital technique.
using the RpfB protein especially in Indonesia. Therefore, Blood collection after immunisation was performed to
this study aimed to construct a recombinant DNA-rpfB obtain serum that was expected to contain antibodies,
vaccine originating from the Beijing strain of M. which were then used in expression testing using
tuberculosis, local to Indonesia, and analyse its immunostaining and Western blot. All mice
expression capability in vitro, as well as the humoral experiments were approved through Ethical Approval
immune response generated, in order to provide No. 984/UN2.F1/ETIK/2016 from Ethical Committee
additional data to develop a new TB vaccine seed. Faculty of Medicine, Universitas Indonesia.

Methods Humoral immune response test in mouse serum

(Western blot). The RpfB protein was obtained from
rpfB gene amplification. Forward and reverse primers the pGEX-rpfB BL21 strain of E. coli (constructed by
were independently designed by using BioEdit version Rukmana et. al., 2014, Submitted) and its expression
3.0 software. The primer pair was designed specifically was induced by the addition of 0.1 mM isopropyl-β-D-
to the open reading frame of rpfB of the M. tuberculosis galactosidase (IPTG). The protein profile was then
Beijing strain. In the forward primer, a HindIII analysed and visualized using 15% SDS-PAGE followed
restriction site was inserted, and in the reverse primer, by staining.
EcoR1 was inserted. The sequences were: rpfB–F, 5′-
CGCAAGCTTATCATGGCG…TTG…...-3′ and rpfB′R, The mouse serum was analysed using Western blotting
5′-CGCGAATTC…GCG………-3′. The unfilled dots to SDS PAGE where GST-RpfB, and GST protein were
show base components that are not provided for patent run. Briefly, The protein samples were transferred from
purposes. the SDS gel using the semi-dry transfer method with a
bottom-to-top sequence of Whatman paper, Hybond-N-
Cloning process of rpfB gene with pcDNA3.1. The Extra membrane, polyacrylamide SDS gel and Whatman
rpfB gene and pcDNA3.1 plasmid were cut using the paper. The transfer process was performed using Trans
EcoR1 (Fermentas) and HindIII (Fermentas) enzymes Blot SD-Semi Dry Electrophoresis Transfer (Biorad) in
and then ligated using T4 DNA ligase (Fermentas). The a transfer buffer (2.5 g glycine, 5.8 g Tris-Base, 200 mL
ligation products were then transformed into the methanol and H2O to a final volume of 1 L), 5 V voltage
Escherichia coli DH5α strain. The success of this and 0.1 A for 2 hAfter the protein was successfully
recombinant plasmid transformation was confirmed by transferred into the membrane, the membrane was then
PCR analysis of a bacterial colony that probably soaked in blocking solution that contained 5% skimmed
contains rpfB gene insert in plasmid, plasmid cutting milk in 1× Tris-buffered saline and Tween 20 (TBST)
with Stu1 restriction enzyme and sequencing. The solution and incubated overnight at 16 °C. The solution
positive colonies were then grown in 10 mL LB was then removed and changed to 1× TBST solution
medium with 100 µg/mL ampicillin overnight at 37 oC, containing 1% skimmed milk and mouse serum diluted
and then 1 mL of culture dissolved in LB medium to 1/1000. As a control, the peroxidase/GST-labelled
contain glycerol 10% for isolate stock. anti-GST antibody (AbCam) was used diluted to 1/5000

Makara J. Health Res. April 2018 | Vol. 22 | No. 1

10 Dian S., et al.

and incubated for 1 h with slow shaking at 2 rpm. times with 1× PBS (5 min each wash), added 200 µL of
Control membranes (anti-GST) were then washed three Nacalai substrate and incubated for 30 mins. The
times with 1× TBST (5 min each wash) at 2 rpm at substrate solution was removed and the cells were
room temperature. On the other hand, the membrane washed with 1× PBS and observed under a microscope
with mouse serum was resoaked in 1× TBST that using a 40× magnification.
contained 1% skimmed milk with immunoglobulin G
(IgG) horseradish peroxidase (HRP) mouse α-goat Results
antibody (Invitrogen) diluted to 1/5000, incubated for 1
h at room temperature and slowly shaken at 2 rpm. Recombinant pcDNA3.1-rpfB construction. rpfB
After that, the membrane was washed three times with gene was successfully amplified in size of 1101 bp using
1× TBST (5 min each wash). Finally, both membranes specific primers with EcoR1 and Hind III restriction
were soaked in TMB substrate (KPL) and incubated enzyme (data not shown), thus it was cloned in to
until the protein band was visible. The membrane was pcDNA3.1 expression plasmid through heat shock method
photographed for documentation purposes. in to competent E. coli DH5α bacteria. Following
transformation, a total 95 colonies were obtained, and
pcDNA3.1-rpfB expression analysis in mammalian 42 colonies were chosen randomly for PCR screening
cells. The pcDNA3.1-rpfB recombinant plasmid using insert and plasmid primers. Four recombinant
transfection into Chinese hamster ovary (CHO-K1) cells plasmid from positive colonies of PCR screening (data
was performed using the cationic lipid method using not shown) were further analysed using StuI restriction
lipofectamine LTX and Plus Reagent (Invitrogen). The enzyme. This enzyme has only one restriction site in
cells were grown in 24-well plates for 24 h in an pcDNA3.1 plasmid, thus recombinant plasmid with
incubator with 5% CO2 CHO-K1at 37 °C in MEM insert will show higher band (6517 bp) than the original
(Sigma) supplemented with 10% foetal bovine serum plasmid (5428 bp) as the result were depicted in Figure
(FBS). Cell growth inside the plate was observed until a 1. Furthermore, 2 out of 4 recombinant plasmids were
minimum of 80% confluence was reached. This cell chosen for sequencing analysis as the result showed that
concentration is optimum condition for transfection there were no mutations or insertions in the whole
process. recombinant DNA construction (sequence data was not
shown due to patent issue).
The transfection was performed in 1.5-mL eppendorf
tubes with 200 µL of OPTIMEM medium (Gibco). Humoral immune response test in mouse serum
First, 500 ng of the recombinant plasmid was added, (Western blot). To asessed the ability of pcDNA3.1-
then 0.3 µL of Plus Reagent was added into the mixture rpfB DNA vaccine to induce humoral immune response,
and incubated for 5 min at room temperature. Then, 1 western blot analysis was performed using pGEX-rpfB
µL of lipofectamine was added and incubated at room expression system and mice immunization sera. As
temperature for 30 min. The medium of the CHO-K1 depicted in Figure 2, native pGEX6p-1 plasmid is not
cells to be transfected was removed and the wells were producing any band in both induced and non-induced
washed three times with OPTIMEM medium. The IPTG (lane 1 and 2). Meanwhile, recombinant pGEX-
plasmid mixture was added to the CHO-K1cells and rpfB plasmid was shown to give positive single protein
incubated with 5% CO2 at 37 °C for 4 h. Afterwards, the band between 50 – 70 kDa, predicted as RpfB-GST
lipofectamine solution was removed and 200 µL of recombinant protein (66 kDa) in both induced and non-
MEM supplemented with 10% of FBS was added into induced IPTG (lane 3 and 4).
the wells and incubated overnight in a CO2 incubator at
37 °C. This result indicated that pcDNA3.1-rpfB DNA vaccine
is able to induce humoral immune response in mice
After 24 hours, the cells were washed three times with which recognize specifically in to RpfB protein since
1×phosphate-buffered serum (PBS) and then dried pcDNA3.1-rpfB DNA vaccine was constructed without
overnight at room temperature. The cells were then any additional tag protein correspond to the absence of
fixed by adding 500 µL of cold absolute ethanol and any band in native pGEX6p-1 lanes. The presence of
incubating at −20 °C for 30 mins. The ethanol was then positive band was shown in pGEX-rpfB non-IPTG
removed and the cells were dried at room temperature. induced (lane 3) may be the result of any basal
The fixed cells were stained by adding serum diluted to expression system that happen inside the cells which
1/100 from immunised mice into 200 µL of 1× PBS and allow the RpfB-GST protein expression. Thus, this
incubating for 1 hour at room temperature. This was expression was highly determined by the presence of
followed by three washes using 1× PBS (5 mins each IPTG inducer in the system as thicker target band
wash). Then, 200 µL of secondary antibody (IgG HRP protein was shown (lane 4). This western blot
mouse α-goat diluted to 1/500 with 1× PBS) was added analysis was also done using anti-GST monoclonal
into the cells and reincubated for 1 hour at room antibodies (Abcam) as control.
temperature. After that, the cells were washed three

Makara J. Health Res. April 2018 | Vol. 22 | No. 1

 Development of a Tuberculosis Vaccine Seed 11

rpfB (1089
rpfB (1089 bp) bp)
GST is a protein from Schistosoma japonicum. It is a
natural protein with a molecular weight of 26 kDa and
StuI (2053) consists of 220 amino acids. In general, GST is used to
pCMV BGH pA F1 ORI
produce a fusion protein, which is attached to the protein

SV40
at the end of the N-terminus.12 Western blot analysis
Ampicillin

ori
pCDNA 3.1
(5428 bp)
using anti-GST monoclonal antibodies resulting similar
band size (range at 50–70 kDa) on lane 3 and 4 and
ColE1 Neomycin
SV40 pA single band at range of 25–30 kDa on lane 2, indicating
the expression of protein fusion of RpfB-GST (data not
shown).

pCMV rpfB BGH pA pcDNA3.1-rpfB expression analysis in mammalian

F1 ORI
cells. The pcDNA3.1-rpfB recombinant plasmid
Ampicillin

StuI (2053)
pCDNA 3.1-rpfB expression was confirmed in a mammalian expression
(6517 bp)
system using CHO-K1 cells and then analysed by
ColE1 SV40 pA Neomycin SV40 ori immunostaining the transfected cells. Immunostaining
was conducted using mouse serum that had been
(a)
immunised with pcDNA3.1-rpfB and confirmed to
contain anti-RpfB antibodies. The expression result
analysis is shown in Figure 3.Figure 3A is a negative
transfection control, where CHO-K1 cells were
plasmid from transfected using native pcDNA3.1 plasmid and stained
restriction with the immunized mice sera resulting no violet stained
cells observed under the microscope. In figure 3B, is a
positive control of transfected CHO-K1 cells with
pcD2ME (recombinant pcDNA3.1 plasmid containing
prM/E dengue virus gene expression system)13 and
stained with dengue patients sera resulting violet stained
cells. On the other hand, pcDNA3.1-rpfB was
transfected in to CHO-K1 cells and stained using
immunized mice sera which also resulting violet stained
(b)
cells (Figure 3C). Violet stained cells which observed is
Figure 1. a). Construction of Recombinant DNA Plasmid. the result of chemical reaction between conjugated
b). Electrophoresis of Stu1 Restriction Result secondary horseradish peroxidase enzyme and
(black arrow). M, Marker for 1 kb; Lane 1, diaminobenzydine substrate, indicating the recombinant
pcDNA3.1 Plasmid; Lane 2, pcDNA3.1 Plasmid rpfB antigen was expressed inside the cells.
Cut by Stu1 Restriction Enzyme; Lanes 3–6,
pcDNA3. 1-rpfB Recombinant Plasmid cut by
Stu1 Restriction Enzyme

M 1 2 3 4

70 kDa 66 kDa
50 kDa A B

C
Figure 3. CHO-K1 Cells Transfected with Recombinant
Plasmid. A, Negative Control (CHO-K1 Cells
Figure 2. Western Blot Result of RpfB Protein. M, Protein Transfected with the Empty pcDNA3.1 Plasmid); B,
Marker; Lane 1, GST without IPTG Induction; Positive Control (CHO-K1 Cells Transfected with
Lane 2, GST with IPTG Induction; Lane 3, Dengue prM-E Recombinant Plasmid); C, CHO-
RpfB-GST without IPTG Induction; Lane 4, K1 Cells Transfected with the pcDNA3.1-rpfB
RpfB-GST with IPTG Induction Recombinant Plasmid

Makara J. Health Res. April 2018 | Vol. 22 | No. 1

12 Dian S., et al.

Discussion Therefore, the seed vaccine resulting from this study

may eliminate the dependence on imported materials.14
TB is an infectious disease caused by M. tuberculosis
bacteria. According to the WHO, until now, TB is one In this study we have developed a novel TB DNA
of the most infectious diseases and has a high mortality vaccine, pcDNA-rpfB, which had been proven to be
rate worldwide.1 One approach to prevent TB is expressed in mammalian cell line and able to induce
vaccination or healthy lifestyle, as well as sanitation humoral antibody response in mice. Similar results were
improvement in public areas. The BCG vaccine is obtained by Lee et al. (2014), who also used the rpfB
currently the only TB vaccine used, although its effec- DNA vaccine approach obtained from Mtb H37Rv
tiveness is diminishing over time and its efficacy differs strain.15 The DNA vaccine used was pcDNA3.1-rpfB
in some areas. In addition to its efficacy problem, the was also injected to mice, meanwhile the antibody
BCG vaccine does not provide protection against latent response was tested by enzyme-linked immunosorbent
TB infection and subsequent infection reactivaton.3,4 assay. From previous study, specific response against
RpfB protein was formed as IgG antibody classes.16
Research into possible new TB vaccine candidates has
been conducted around the world, especially where the The anti-RpfB antibody was formed as a humoral
host immune response to TB is known. Most of M. immune response generated by mice as a result of the
tuberculosis infection is manifested as latent infection, rpfB DNA vaccine. Theoretically, after the DNA
where bacteria is in dormancy and regulates survival vaccine was injected into the tissue, the DNA plasmid
genes within its genome to adapt the host internal would replicate autonomously to produce foreign
environment and to evade host immune system. protein that was antigenic. The foreign protein then
Resuscitation-promoting factor (Rpf) is one gene cluster induced B cells to produce antibodies for the antigen.
which is well known to play a role in M.tb dormancy. The DNA vaccine not only stimulated the humoral
The proteins encoded by the rpf genes are important to immune response through antibody formation, but also
the survival of the bacteria in the latent condition.5,6 stimulated a cellular immune response through T-cell
According to previous research, the RpfB protein has activation. This provided an advantage to the infections
the highest biological and immunological characteristics caused by intracellular pathogen because cytotoxic T
among other proteins of the Rpf family.7 This makes the cells could kill infected cells or activate cytokine
rpfB protein is an appropriate target to be developed as production and, therefore, stop the infection spreading.17
an antigen in designing a TB vaccine so that it can elicit
an immune response to the latent Mtb infection, enabling Mammalian cells have sophisticated expression systems,
dormant bacteria to be eliminated and subsequent TB such as chaperones, attachment sites, apparatus for
reactivation to be avoided.8 protein secretion and post-translational modifications, to
help proteins to fold precisely.15 Previous research has
DNA-based vaccines are currently under study. The advan- demonstrated that the RpfB protein of M. tuberculosis
tages of this vaccine approach is only containing a foreign was successfully expressed in prokaryotic system such
DNA substance which relatively safe, can induce both as E. coli, and isolated and purified.18 As a prokaryotic
humoral and cellular immune response, easy to design expression system, E. coli cannot perform glycosylation,
and to be handled for their stability at room temperature.9 which is important for post-translation modification,
This study aimed to target a gene from the rpf family of especially for protein that exists inside the cell membrane,
the M. tuberculosis Beijing strain isolated in Indonesia as RpfB protein is positioned inside the cell19,20 as well
to produce an rpfB single DNA vaccine. This was tested as being secreted out of cells.15 Therefore, by using
in male Balb/C mice to observe the humoral immunity mammalian expression system, we hope that our
response as well as the rpfB gene expression in a recombinant protein product can be folded and modified
mammalian expression system by transfection. correctly, better than prokaryotic expression system in
order to obtain better protein antigenicity and
The M. tuberculosis Beijing strain used in this study immunogenicity.
was obtained from the Microbiology Department culture
collection of Medical Faculty, Universitas Indonesia. M. Conclusions
tuberculosis Beijing strain itself is the most dominant
TB strain in Asia, especially Indonesia, and has We concluded that the rpfB gene from the Beijing strain
characteristics such as it is a more virulent strain and of M. tuberculosis can induce a humoral immune response
resistant to multiple anti-TB medicines. This local in mice injected with the pcDNA3.1-rpfB recombinant
isolate utilisation is one approach to support the plasmid and that the protein was successfully expressed
government policy about independently supplying raw in the mammalian cells. We hope that these preliminary
drug materials and vaccines, as stated in the Strategic data can be used in future research as an approach to
Plan Development of pharmaceutical industry and develop a TB vaccine seed using the Beijing strain from
health equipment of the Ministry of Health, 2016. the local isolate.

Makara J. Health Res. April 2018 | Vol. 22 | No. 1

 Development of a Tuberculosis Vaccine Seed 13

Acknowledgement tuberculosis resuscitaion-promoting factors as antigens in

novel tuberculosis sub-unit vaccines. Microbes Infect.
None. 2012;14:86-95.
10. Tufariello JM, Mi K, Xu J, Manabe YC, Kesavan AK,
Drumm J, et al. Deletion of the mycobacterium
Funding tuberculosis resuscitation-promoting factor Rv1009 gene
results in delayed reactivation from chronic tuberculosis.
This study is funded by Kemenristekdikti through Hibah Infect Immun. 2006;74:2985-95.
Kompetensi 2015-2016 fiscal year. 11. Kinney RM, Huang C. Development of new vaccination
against dengue fever and Japanese encephalitis.
Conflict of Interest Statement Intervirology. 2001;44:176-97.
12. Harper S, Speicher DW. Purification of proteins fused to
glutathione S-tranferase. Methods Mol Biol.
None to declare.
2011;681:259-80.
13. Putri DH, Sudiro TM, Yunita R, Jaya UA, Dewi BE,
References Sjatha F, Konishi E, Hotta H, Sudarmono P. Immuno-
genicity of a candidate DNA vaccine based on the prM/E
1. WHO. Global tuberculosis report. [Internet]. 2017 [cited genes of a dengue type 2 virus Cosmopolitan genotype.
2017 Jan 6]. Available from: http://www.who.int/ Jpn J Infect Dis. 2015;68:357-63.
mediacentre/factsheets/fs104/en/ 14. Kementerian kesehatan. Setditjen Kefarmasian dan Alkes.
2. Velayati AA, Abeel T, Shea T, Zhavnerko GK, Birren B, Buletin Informasi Kefarmasian dan Alat Kesehatan.
Cassell GH, et al.Populations of latent Mycobacterium Upaya kemandirian produksi bahan baku obat Indonesia.
tuberculosis lack a cell wall: Isolation, visualization, and 2016. [In Indonesian]
whole-genome characterization. Int J Mycobacteriol. 15. Lee J, Kim J, Lee J, Shin SJ, Shin EC. DNA immunization
2016;5;66-73. of mycobacterium tuberculosis resuscitation-promoting
3. Rubio-Reyes P, Parlane NA, Buddle BM, Wedlock DN, factor B elicits polyfunctional CD8+ T cell responses.
Rehm BHA. Immunological properties and protective Clin Exp Vaccine Res. 2014;3:235-43.
efficacy of a single mycobacterial antigen displayed o 16. Burhannudin. Isolasi, purifikasi dan studi imunogenisitas
polyhydroxybutyrate beads. Microb Biotechnol. 2017; protein rekombinan resuscitaion promoting factor B
10:1434-40. (RpfB) Mycobacterium tuberculosis secara invitro pada
4. Im EJ, Saubi N, Virgili G, Sander C, Teoh D, Gatell JM, splenosit mencit. [Thesis]. Depok. Universitas Indonesia;
et al. Vaccine Platform for prevention of tuberculosis and 2015.
mother-to-child transmission of human immuno-deficiency 17. Xia W, Peter B, John M, Patrick PJ, Warren M, Ying Z,
virus type 1 through breastfeeding. J Virol. 2007;81:9408–18. et al. High level of protein expression using different
5. Svenson S, Källenius G, Pawlowski A, Hamasur B. mammalian CMV promoters in several cell lines. Sci
Towards new tuberculosis vaccines. Hum Vaccines. Direct. 2006;45:115-24.
2010;6:309–17. 18. SnapGene. Plasmid files [Internet]. 2017 [cited 2017 Jan
6. Thaiss CA, Kaufmann SH. Review, toward of novel 5]. Available from: https://www.snapgene.com/resources/
vaccines against tuberculosis: Current hopes and plasmid_files/basic_cloning_vectors/pcDNA3.1(+)/
obstacles. Yale J Biol Med. 2010;83:209–15. 19. Dyson MR. Fundamentals of expression in mammalian
7. Russell-Goldman E, Xu J, Wang X, Chan J, Tufariello cells. In: Vega M, editor. Advanced Technologies for
JM. A mycobacterium tuberculosis Rpf double-knockout Protein Complex Production and Characterization.
strain exhibits profound defects in reactivation from Switzerland: Springer; 2016. p. 217-22.
chronic tuberculosis and innate immunity phenotypes. 20. Bavaro T, Tengattini T, Piubelli L, Mangione F,
Infect Immun. 2008;76:4269-81. Bernardini R, Monzillo V, et al. Glycosylation of
8. Fan A, Jian W, Shi C, Ma Y, Wang L, Peng D, et al. recombinant antigenic proteins from mycobacterium
Production and characterization of monoclonal antibody tuberculosis: In silico prediction of protein epitopes and
against Mycobacterium tuberculosis RpfB domain. Ex Vivo Biological Evaluation of New Semi-Synthetic
Hybridoma. 2010;29:327-32. Glycoconjugates. Molecules. 2017;22:1081-97.
9. Romano M, Aryan E, Korf H, Bruffaerts N, Franken
CLMC, Ottenhoff THM, et al. Potential of mycobacterium

Makara J. Health Res. April 2018 | Vol. 22 | No. 1