Chikungunya is one of the current infectious, re-emerging diseases. Chikungunya virus (CHIKV) infection can cause some non-specific symptoms such as fever, arthralgia, and myalgia, similar to dengue and zika virus infection. The similarity of the symptoms frequently makes the diagnosis inaccurate. Therefore, a rapid and accurate diagnostic method such as an antibody-based Rapid Diagnostic Test (RDT) is needed to prevent a late or inaccurate diagnosis that can cause prolonged effects of infection. This study aims to obtain a recombinant clone colony of P. pastoris X-33 as an initial step for antibody-based RDT kit development. This study includes the transformation and the Mut+ phenotype confirmation of recombinant clones. The recombinant plasmid pPICZaA-E2 was transformed and integrated into P. pastoris X-33 genome using the electroporation method. After screening process through genome PCR, four positive recombinant clones colonies were analyzed in minimal dextrose and methanol media for Mut+ confirmation. Visualization of PCR colony shows two bands were formed in the size of 2.200 bp corresponding to the P. pastoris genome’s AOX1 gene and 1.848 bp corresponding to recombinant plasmid’s AOX1 gene that indicates the recombinant plasmid pPICZaA-E2 was integrated into the genome. All of the recombinant clones colonies grew well in minimal dextrose and minimal methanol media. Based on the results, it can be predicted that the Mut+ phenotype of P. pastoris recombinant clone was successfully obtained. However, gene sequencing must be done as a further step to confirm the presence of the recombinant DNA and the AOX1 gene in P. pastoris.

1.
A. M.
Powers
,
A. C.
Brault
,
R. B.
Tesh
and
S. C.
Weaver
,
J. Gen. Virol.
81
,
471
479
(
2000
).
2.
E. S.
Paixao
,
M. G.
Teixeira
and
L. C.
Rodrigues
,
BMJ Glob. Health
3
,
1
6
(
2017
).
3.
S. K.
Mardekian
and
A. L.
Roberts
,
BioMed Res. Inter.
15
,
1
8
(
2015
).
4.
T.
Okabayashi
 et al.,
J. Clin. Microbiol.
53
,
382
388
(
2015
).
5.
E.
Burdino
,
G.
Calleri
,
P.
Caramello
and
V.
Ghisetti
,
Emerg. Infect. Dis.
22
,
1837
1839
(
2016
).
6.
7.
S. W.
Metz
and
G. P.
Pijlman
, “Function of Chikungunya Virus Structural Proteins,” in
Chikungunya Virus
, edited by
C.
Okeoma
(
Sprinegr
,
Cham, New York
,
2016
) pp.
63
74
.
8.
J.
Kim
 et al.,
Virol. Sin.
34
,
563
571
(
2019
).
9.
B.
Cho
 et al.,
Yonsei Med. J.
49
,
828
835
(
2008
).
10.
A.
Verma
 et al.,
J. Virol. Methods
235
,
73
79
(
2016
).
11.
P.
Tiwari
 et al.,
Mol. Biotechnol.
46
,
80
89
(
2010
).
12.
S. W.
Metz
 et al.,
Virol. J.
8
,
353
(
2011
).
13.
J. L.
Cereghino
and
J. M.
Cregg
,
FEMS Microbiol. Rev.
24
,
45
66
(
2000
).
14.
M. W.
Nasser
,
V.
Pooja
.,
M. Z.
Abdin
and
S. K.
Jakin
,
IJBT
2
,
477
493
(
2002
).
15.
I. A.
Pla
 et al.,
Biotechnol. Prog.
22
,
881
888
(
2006
).
16.
J. M.
Cregg
,
Pichia Protocols
(
Humana Press
,
New Jersey
,
2007
), p.
261
.
17.
Zymo Research
,
Zymoprep Manual Cat No. D2001
available at https://files.zymoresearch.com/protocols/-_d2001_zymoprep_yeast_plasmid_miniprep_i.pdf.
18.
Invitrogen
(
2010
),
Pichia Expression Manual Cat No. K1740-01
, available at http://tools.thermofisher.com-/content/sfs/manuals/easyselect_man.pdf.
19.
S.
Linder
,
M.
Schliwa
and
E.
Kube-Granderath
,
Biotechniques
20
,
980
982
(
1996
).
20.
O-J.
Burrowes
,
G.
Diamond
and
T-C.
Lee
,
J. Biomed. Biotechnol.
4
,
374
384
(
2005
).
This content is only available via PDF.
You do not currently have access to this content.