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Procedia Chemistry 18 (2016) 237 245

Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and Structural Biology
2015 Conference, MCLS 2015

Immunogenicity and Specificity of Anti recombinant Protein Fim-

C-Salmonella typhimurium Antibody as a Model to Develop
Typhoid Vaccine
Muktiningsih Nurjayadia*, Dea Apriyania, Umar Hasana, Imam Santosoa, Fera
Kurniadewia, Irma Ratna Kartikaa, Kurnia Agustinic, Fernita Puspasarib, Dessy Nataliab,
Wibowo Mangunwardoyod
a
Chemistry Department, Faculty of Mathematics and Natural Sciences, Universitas Negeri Jakarta, Jakarta 13220, Indonesia
b
Biochemistry Division, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Indonesia
c
Biofarmaka Research Laboratory LAPTIAP BBPT Serpong Indonesia
d
Biology Department, Faculty of Mathematics and Sciences, Universitas Indonesia, Depok, 16424, Indonesia

Abstract
Salmonella typhimurium, that cause typhoid fever in mice was used as a model to study typhoid fever in humans. This study is
aimed to determine the immunogenicity of recombinant protein of Fim-C-S. typhimurium and specificity of antibody anti-
recombinant protein of Fim-C-S. typhimurium in ddY mice. The immunogenicity studies by ELISA indicated that recombinant
protein of Fim-C-S. typhimurium evokes strong immune response in ddY mice, particularly in experiment group. The specificity
evaluated by Western Immunobloting technique indicated that the antibody anti-recombinant protein of Fim-C-S. typhimurium
can detect the 31 kDa of recombinant protein of Fim-C-S. typhimurium as an antigen. This data reported that recombinant protein
of Fim-C-S. typhimurium is immunogenic and the antibody anti-recombinant protein of Fim-C-S. typhimurium is specific. The
results of this study can be used as a model to develop recombinant vaccine candidate against typhoid.

2016
2015Published by Elsevier
The Authors. B.V. by
Published ThisElsevier
is an open
B.V.access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases,
Peer-review
Biochemistry under
andresponsibility of the organizing
Structural Biology 2015 (MCLS committee
2015).of the Molecular and Cellular Life Sciences: Infectious Diseases,
Biochemistry and Structural Biology 2015 (MCLS 2015)
Keywords: Salmonella typhimurium; typhoid fever; recombinant protein Fim-C-Salmonella typhimurium antibody; recombinant vaccine

* Corresponding author. Tel.: +62-21-4896669; +62-021-5465245; fax: +62-21-4894909, HP : +62-81511127784/+62-81517249667.

E-mail address: muktiningsih@unj.ac.id

1876-6196 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the organizing committee of the Molecular and Cellular Life Sciences: Infectious Diseases, Biochemistry and
Structural Biology 2015 (MCLS 2015)
doi:10.1016/j.proche.2016.01.037
238 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245

1. Introduction

Typhoid fever, caused by Salmonella typhi (S. typhi) bacteria, is a major cause of illness and death, particularly
among children, in developing countries. By the year 2000, it is estimated 22 million cases of typhoid fever and
200.000 deaths occurring worldwide3,4,20. However, to study the pathogenesis of S. typhi in human is restricted.
Thus, the urgency to find a pathogen with similar symptoms should be carried out. Salmonella typhimurium (S.
typhimurium), that cause typhoid fever in mice and has similar symptoms to typhoid fever in human, are used as a
model.
Salmonella typhimurium are pathogenic, Gram negative, facultative anaerobic bacilli which have flagels and
fimbriae and live in human and animal intestinal tracks. The organisms stimulate different effects in different host
cells. Those cause gastroenteritis in humans, while also typhoid fever in mice. Therefore, S. typhimurium in infected
mice are used as a model to study the pathogenesis of S. typhi in humans17,21. Typhoid fever is overcome by
vaccines. Two available vaccines against typhoid, Ty21a (oral vaccine) and Vi CPS (injection vaccine), are
produced from attenuated strains of S. typhi. However, those vaccines provide moderate protection against typhoid,
especially in hyper-endemic areas5,8. In Indonesia, effectivity of protection against typhoid is 33%-53% within three
years lower than other countries21. Therefore, it has stirred an urgency to develop better and efficient typhoid
vaccines.
Recombinant protein vaccine is one of the alternatives against typhoid. There are advantages of recombinant
protein vaccine, such as higher protection for patients, higher purity, more specific, and allowing the production of
large quantities14,21. The recombinant subunit approaches are being used for the development of new vaccines and
improvement of already existing vaccines2. Many recombinant vaccines are now being tested in preclinical and
clinical trials, and some recombinant vaccines are already on the market. Some recombinant vaccine showed at
Table.1.
Table 1. Selected examples of recombinant vaccines currently approved in the US or EU

Vaccine Description Application Company Approved

Recombivax rHBsAg produced in S. cerevisiae Hepatitis B prevention Merck 1986 (US)
Comvax Combination vaccine, containing Vaccination of infants against H. Merck 1996 (US)
rHBsAg produced in S. cerevisiae influenzae type B and hepatitis B
as one component
Tritanrix-HB Combination vaccine, containing Vaccination against hepatitis B, diphteria, SmithKline 1996 (EU)
rHBsAg produced in S. cerevisiae tetanus, and pertussis Beecham
as one component
Twinrix, adult Combination vaccine, containing Immunization against hepatitis A and B SmithKline 1996 (EU)
and pediatric rHBsAg produced in S. cerevisiae Beecham (adult)
forms as one component 1997 (EU)
(pediatric)
Primavax Combination vaccine, containing Immunization against diphteria, tetanus, Pasteur 1998 (EU)
rHBsAg produced in S. cerevisiae and hepatitis B Merieux
as one component MSD
Procomvax Combination vaccine, containing Immunization against H. influenzae type B Pasteur 1999 (EU)
rHBsAg produced in S. cerevisiae and hepatitis B Merieux
as one component MSD
Lymerix rOspA, a surface lipoprotein of B. Lyme disease vaccine SmithKline 1998 (US)
burgdorferi, produced in E. coli Beecham

The previous research in 2007-2009, we found thirteen potential S. typhi protein, one of them is Fim-C S. typhi,
using 2D and MALDI TOFF17. In parallel we also study Fim-C S. typhimurium protein as a model. The Fim-C S.
typhimurium protein as well known outer membrane protein. References analysis indicated that the outer membrane
proteins of Salmonella have strong immunogenic potential, this characteristic is supporting for development of
vaccine. The use of recombinant proteins allows the targeting of immune responses focused against few protective
antigens was studied by Nascimento and Leite [2012]. There are a variety of expression systems with different
advantages for recombinant protein, such as allowing the production of large quantities of proteins depending on the
required characteristics, a high purity, specific and easy to handling11. In parallel research, we have been developing
of recombinant protein of Fim-C-S. typhi as vaccine candidate against typhoid13. Earlier study carried out in our
 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245 239

laboratory, we have done isolated, cloned, expressed of fim- C- S. typhimurium gene to be recombinant protein of
fim- C- S. typhimurium, and purified and characterize of recombinant protein of Fim- C- S. typhimurium1,6,17,18,19.
This study is aimed to determine the immunogenicity of recombinant protein of Fim C S. typhimurium and
specificity of anti recombinant protein of Fim-C-S. typhimurium antibody in ddY mice. The research is very
important as model/basic on developing recombinant typhoid vaccine for human.

2. Methods

2.1. Antigen

Antigen used in this experiment is recombinant protein Fim-C-S. typhimurium in the form inclusion bodies.
Antigen of Fim-C-S. typhimurium was produced in the previous research using recombinant DNA
technology1,5,16,17,18. The procedure for production of recombinant protein of Fim-C-S. typhimurium as follow: (1)
isolation of the genome S. typhimurium LT2 by Wizard kit28, (2) amplification of fim-C-S. typhimurium gene 0.7 kb
size by PCR method (3) cloning gene of fim-C-S. typhimurium 0.7 kb on vector cloning pGMTeasy kit22, which
constructed recombinant plasmid pGEM-fim-C-S. typhimurum, (4) subcloning gene fim-C-S. typhimurium from
plasmid recombinant pGEM-fim-C-S. typhimurum to expression vector pET30A [pET system, Invitogen, 2012] to
produced plasmid recombinant pET30A- fim-C-S. typhimurum, (5) Expression of recombinant protein of Fim-C-S.
typhimurum, (6) isolation and purification of recombinant protein of Fim-C-S. typhimurum in inclusion bodies form
by Hispur kit26.
Preparation of antigen for imunization as follow as: the amount of 20-60 Pg recombinant protein Fim-C-S.
typhimurium in inclusion bodies form resulted on the purification disolved with buffer PBS 1x on Eppendorf 1,5
mL the total volume was 100-500 PL. Addition with Freunds complete adjuvant (FCA) or Freunds incomplete
adjuvant (FIA) with equal comparation 1:1. Homogenation by vortexing up to the white color appear16.

2.2. Animal Tested

Production of antibody anti recombinant protein of Fim-C- S. typhimurium was conducted in ddY strains, 6-8
weeks old, 17-24 gr male mice. Antibody of anti recombinant protein of Fim-C-S. typhimurium was obtained from
36 mice that have been categorized into two major groups, experiment group and control group. Each of the
experiment groups were divided into two sub groups, the group that was immunized by mixed Fim-C S.
typhimurium recombinant protein and Freund complete/incomplete Adjuvant (KP-1) and the group that was
immunized only by Fim-C protein dilution in PBS 1x (KP-2). The control groups divided into two sub groups, the
first group that was immunized by Freund Complete/Incomplete Adjuvant (KS-1) and the other group that was
immunized by Phosphate Buffer Saline or PBS 1x (KS-2). Each of the groups consisted of 8 mice. Four other mice
were treated as control in each group. They were categorized into normal group (KN), without immunized11,16.

2.3. Immunization

Immunization was conducted as many as four times in day 1, day 9, day 17, and day 25. Each immunization was
performed in 1-2 injections using 20 g (dose 1), 40 g (dose 2) and 60 g (dose 3 and 4) on the dorsal near head for
experiment groups11.

2.4. Test Bleeds

Test bleeds were done from mices eyes sinus orbitalis. Pre-immune serum was taken before the treatment began
(day 0). Continuously, the test bleed was taken on day 8 (test bleed 1), day 16 (test bleed 2), day 24 (test bleed 3),
and day 32 (test bleed 4). 500 PL bloods from eyes sinus orbitalis was incubated at 37oC for 1 hour and was
centrifuged at 5000 g for 10 minutes at 4oC. The serum was removed from the cell pallet and inserted to sterile
Eppendorf tube. The serum then was frozen at -20oC. The yield was approximately 100-200 PL11.
240 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245

2.5. Enzyme Linked Immuno Sorbent Assay (ELISA)

As much as 100 ng and 300 ng of Fim-C S. typhimurium recombinant protein were used as antigen in ELISA.
Antibody of anti recombinant protein of Fim-C-S. typhimurium in ELISA was diluted 100 times. Amount of 50 L
antigen diluted in PBS was incubated in each well micro titer plate at room temperature. Each well was washed
three times with PBS contained by 1 mM MgCl2 and 0.05% (v/v) Tween-20. As much as 150 L 5% blotto (5 g
skimmed milk in 100 mL PBS) was added in each well, and then micro titer plate was incubated at 37C for one
hour. Micro titer plate was washed three times with PBS. The 50 L mice serum from the fourth test bleeds with
1000 times dilution concentration was added in each well and incubated at 37C for one hour. The plate was washed
three times with PBS. The 50 L labeled secondary antibody (anti IgG-mice-HRP diluted 5000 times)17 was added
to plate, and then incubated at 37C for one and a half hour. Micro titer plate well was washed with washing buffer
for three times. 50 L substrate solution TMB (3,3,5,5-tetramethylbenzidine, Thermo Scientific) 25,29 was added in
each well and incubated at at 37C for one hour. The color formation reaction was stopped by adding 50 L H2SO4.
After the incubation, it was measured the absorbance value by ELISA-Reader in 450 nm wave length. The result
was the absorbance value in each well8,11,16.

2.6. Specificity Test by Western Immunobloting.

Serological test antibody of anti recombinant protein of Fim-C-S. typhimurium against antigen of recombinant
protein Fim-C-S. typhimurium was performed by Western immunoblotting method. In the previous research
characterization of 31 kDa Fim-C S. typhimurium protein was performed6. The result of protein separating by SDS-
PAGE gel was transferred to nitrocellulose membrane using Western immunoblotting apparatus. Protein transfer
process was taken at 4C 200 V in 2-3 hours. Membrane was blocked with skimmed milk to avoid interaction
between primary and secondary antibody. Primary antibody added to membrane was the serum from the fourth test
bleed with 1000 times dilution concentration8,11.

3. Results and discussion

3.1. Immunogenicity Test of Recombinant Protein of Fim-C-S. typhimurium as Antigen

Immunogenicity is the ability of a particular substance, such as an antigen or epitope, to provoke an immune
response in the body of a human or animal. In other words, immunogenicity is the ability to induce a humoral and/or
cell mediated immune response9,11. To evaluate the immunogenicity of recombinant protein of Fim-C S.
typhimurium, the pure recombinant protein described above was prepared for immunization experiment. The ddY
mice were immunized with the antigen formulated with Freunds complete/incomplete adjuvants. Immunization of
mice with increasing protein doses (20 g, 40 g, 60 g, and 60 g) indicated that a 60 g dose gave the highest
anti-Fim-C-S. typhimurium antibody response (Table 2 and Fig. 2).
To compare the immunogenicity of recombinant protein of Fim-C S. typhimurium, in this research the ddY mice
divided in two groups, they are experiment and control group as describe above. The antibody response showed with
increasing absorbance value from ELISA process. As we known the increasing absorbance value shows interaction
between recombinant protein of Fim-C S. typhimurium as antigen and antibody anti recombinant protein of Fim-C-
S. typhimurium. Those interaction showed with the link of the antigen, primary antibody (anti recombinant protein
of Fim-C-S. typhimurium antibody) and secondary antibody anti-mice conjugated with Horse Radish Peroxidase
(HRP) enzyme. Therefore HRP bonded on secondary antibody will oxidize the substrate TMB (3,3,5,5-
Tetramethylbenzidine) became 3,3',5,5'-tetramethylbenzidine diimine.
The resulting diimine causes the solution to take on a blue color, and this color change can be observed on a
spectrophotometer at a wavelength of 650 nm. The reaction can be stopped by addition of acid or another stopper
reagent. Using sulfuric acid turns TMB to be yellow. The color may be read at 450 nm and a higher antibody titre
produced the thicker colour appear. The oxidation reaction of changing 3,3,5,5-tetramethylbenzidine became
3,3',5,5'-tetramethylbenzidinediimine showed at Fig.124,29.
 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245 241

Fig. 1. Oxidation reaction of 3,3,5,5-tetramethylbenzidine became 3,3',5,5'-tetramethylbenzidine diimine. The TMB substrate can act as a
hydrogen donor for the reduction of hydrogen peroxide to water by peroxidase enzymes such as horseradish peroxidase [Wikipedia.org; Sigma
Aldrich Catalogue]

Antigen concentrations in ELISA test were 100 ng and 300 ng. In the experiment group, each of test bleed
produced a higher absorbance in 300 ng antigen concentration. The higher antigen concentration bound higher
amount of antibody. Antigen-antibody interaction, or antigen-antibody reaction, it is a specific chemical interaction
between antibodies produced by B cells of the white blood cells and antigens during immune reaction. It is the
fundamental reaction in the body by which the body is protected from complex foreign molecules, such as
pathogens and their chemical toxins. In the blood, the antigens are specifically and with high affinity bound by
antibodies to form an antigen-antibody complex. The immune complex is then transported to cellular systems where
it can be destroyed or deactivated18.
Table 2 and Fig. 2 show the amount of antibody titer in each test bleed from experiment and control groups.
Antibody titer in experiment group 1 (KP-1) and experiment group 2 (KP-2) increased in every test bleeds, that
showed the increasing antibody response against the antigen. Antibody titer of control group 1 (KS-1, immunized by
adjuvant) and control group 2 (KS-2, immunized by PBS) continuously did not perform the increasing antibody
response. Those results showed that Fim-C S. typhimurium protein has satisfying as immunogenicity.
Table 2. Absorbance value of antibody anti Fim-C S. typhimurium in all groups with 100 ng and 300 ng antigen concentration
Experiment Experiment Control Control
Test Bleed Group 1 (KP-1) Group 2 (KP-2) Group 1(KS-1) Group 2 (KS-2)
100 ng 300 ng 100 ng 300 ng 100 ng 300 ng 100 ng 300 ng
Pre-immune serum 0.01550 0.01400 0.01225 0.01275 0.02150 0.02500 0.03150 0.02550
(test bleed-0)
0.01725 0.01250 0.06875 0.01075 0.02150 0.02300 0.01100 0.02450
1st Test Bleed
nd 0.05725 0.13650 0.03250 0.04200 0.01900 0.02250 0.01150 0.01100
2 Test Bleed
rd 0.20800 0.27700 0.03450 0.07700 0.00900 0.01300 0.01100 0.01650
3 Test Bleed
0.21575 0.32575 0.05950 0.14975 0.01150 0.02300 0.00400 0.00500
4th Test Bleed

The increasing of antibody titer of experiment group 1 (immunized by antigen Fim-C S. typhimurium +CA/FIA
adjuvants (KP-1)) was higher than antibody titer of experiment group 2 (immunized by antigen Fim-C S.
typhimurium + PBS (KP-2)). Those proved that adjuvants can stimulate strong, prolonged responses and protect the
antigen from releasing faster. As we known adjuvants are nonspecific stimulators immune response. The judicious
use adjuvants are essential to induce a strong antibody response to soluble antigens11. Perrie et al, 2008 also reported
the use of adjuvant systems have proven to enhance the immunogenicity of these sub-unit vaccines through
protection (i.e. preventing degradation of the antigen in vivo) and enhanced targeting of these antigens to
professional antigen-presenting cells. However, the experiment group 2 (KP-2) also showed antibody response with
the increasing of antibody titer in each test bleed. Thats means the recombinant protein of Fim-C-S. typhimurium
have a good immunogenicity, because those protein can induce respon immune without adding the adjuvant. So its
very potential as vaccine candidate.
242 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245

The comparison between experiment group 1 (KP-1) and control group 1 (KS-1) showed significant difference to
give response immune. FCA/FIA Adjuvant given alone to the control groups did not affect the production of
antibody. Similar results given by the experiment group 2 (KP-2) and control group 2 (KS-2). The result showed
that only recombinant protein of Fim-C S. typhimurium antigen can induced the increasing of antibody titer. The
result also showed that Fim-C S. typhimurium antigen can respond to specific antibody, while PBS as a solvent and
adjuvant alone does not have immunogenic properties. Compare with other recombinant protein that showed in
Table 1, mostly the recombinant protein with conjugate, however recombinant protein of Fim. C S. typhimurium
even without conjugate can produce a good immune respond That showed those protein have good characteristic as
antigen and vaccine candidate.

Fig. 2. The comparison analysis of antibody titer in all groups. The blue line shows the increasing of antibody in experiment group 1 (KP-1). The
red line shows the increasing of antibody in experiment group 2 (KP-2). The green line shows antibody titer in control group 1 (KS-1). The
purple line shows antibody titer in control group 2 (KS-2). The comparison was performed in 300 ng antigen concentration and 100 times dilution
of antibody anti recombinant protein of Fim-C S. typhimurium.

3.2. Specificity test of anti-Fim-C S. typhimurium antibody

The results of specificity test of anti Fim-C S. typhimurium antibody by Western Immunoblotting showed
interaction between Fim-C antigen and anti-Fim-C S. typhimurium antibody. The interaction was made by the
formation of band with brown color on 31 KDa nitrocellulose membrane, same size as antigen Fim-C S.
typhimurium protein.
The band with brown color was the results of oxidation reaction with peroxides substances (H2O2), which
catalyzed by Horse Radish Peroxidase (HRP) enzyme. The Horse Radish Peroxidase (HRP) enzyme changed DAB
(3,3'-Diaminobenzidine or 3,3',4,4'-Biphenyltetramine) substrate to Quinone Iminium substance which is radical
properties5.
Afterward, the Quinone Iminium substance induced the occurrence of polymerization reaction to form polymer
substance with bigger molecular size and produce precipitate with brown color17. The reaction mechanism to
produce precipitate in brown color with western Immunoblotting was showed at Fig. 3. The specificity test reported
in this research was conducted for experiment group and control group. The condition of antigen-antibody
interaction was respectively as follow (1) 3 g antigen concentration, (2) 100 times dilution of anti recombinant
protein of Fim-C S. typhimurium antibody, and (3) 5000 times dilution of secondary antibody anti-mice.
 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245 243

Fig. 3. Transformation of DAB Substrate to precipitate with brown color by Western Immunoblotting. DAB (3,3'-Diaminobenzidine or 3,3',4,4'-
Biphenyltetramine) donated electron to HRP which was catalyzed from oxidation reaction of peroxides (H2O2) [ Bio-Rad, Laboratories. Inc,
2012]

The Western Immunoblotting showed that the experiment group-1 (KP-1) and experiment group-2 (KP-2)
produced antibody respond against antigen in nitrocellulose membrane. Meanwhile control group 1 and control
group 2 did not produce the antibody. The results of testing were shown in Fig. 4.

Fig. 4. Results of SDS PAGE (blue) and Western Immunoblotting (band with brown color) in all groups. The interaction of antigen-antibody of
Fim-C S. typhimurium were identified by the present of band with brown color at column A and column B. Column A: result from SDS PAGE
and Western Immunoblotting KP-1 group. The column B: result from SDS PAGE and Western Immunoblotting KP-2 group. The column C:
result from SDS PAGE and Western Immunoblotting KS-1 group. The column D: result from SDS PAGE and Western Immunoblotting KS-2
group
244 Muktiningsih Nurjayadi et al. / Procedia Chemistry 18 (2016) 237 245

The result showed that recombinant protein of Fim-C S. typhimurium antigens can induce the formation of anti
recombinant protein of Fim-C specific antibodies, proved by the brown band in membrane nitrocellulose.
Meanwhile the control group which not gets immunized by Fim-C antigen gave negative results. Based on the
results we can describe thats only experiment group 1 and 2 which produce the immune response. As we know
there are several types of antibodies and antigens, and each antibody is capable of binding only to a specific antigen.
The specificity of the binding is due to specific chemical constitution of each antibody. The antigenic determinant or
epitope is recognized by the paratope of antibody, situated at the variable region of the polypeptide chain. The
variable region in turn has hyper-variable regions which are unique amino acid sequences in each antibody.
Antigens are bound to antibodies through weak and noncovalent bonds such as electrostatic interactions, hydrogen
bonds, Van der Waals forces, and hydrophobic interactions18.
The use of adjuvant systems have proven to enhance the immunogenicity of these sub-unit vaccines through
protection (i.e. preventing degradation of the antigen in vivo) and enhanced targeting of these antigens to
professional antigen-presenting cells. Understanding of the immunological implications of the related disease will
enable validation for the design and development of potential adjuvant systems. Novel adjuvant research involves
the combination of both pharmaceutical analysis accompanied by detailed immunological investigations, whereby,
pharmaceutically designed adjuvants are driven by an increased understanding of mechanisms of adjuvant activity,
largely facilitated by description of highly specific innate immune recognition of components usually associated
with the presence of invading bacteria or virus. The majority of pharmaceutical based adjuvants currently being
investigated are particulate based delivery systems, such as liposome formulations11. In this research we used the
FCA/FIA, in further research we will develop for other safely adjuvant for human.

4. Conclusion
The recombinant protein of Fim-C-S. typhimurium as antigen can induce the production of anti recombinant
protein of Fim-C-S. typhimurium antibodies in ddY mice. The serological test with Western Immunoblotting showed
that recombinant protein of Fim-C S. typhimurium as antigen can interact with specific anti recombinant protein of
Fim-C S. typhimurium antibodies. The recombinant protein of Fim-C S. typhimurium has good immunogenicity and
its antibodies are specific. Therefore, it is a significant model in the development of typhoid fever vaccine candidate.

Acknowledgements
The research was done under the funding support from DP2M DIKTI through National Strategic Research Grant
in 2012-2013 and Fundamental Research Grant in 2013-2014. Our gratitude to UNJ Rector for the given opportunity
to do research; leaders and staffs of research institution that has facilitated all research proposals on S. typhimurium
and published the results; Dr. Dessy Natalia and Dr, Fernita Puspasari from ITB Bandung and Dr. Kurnia Agustini
from LAPTIAB BPPT Serpong for collaboration.

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