In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b
In silico analysis of the DNA encoding singlechain Fv antibodies (ScFv) specific to the human recombinant interferon β1b and α2b (rhIFNβ1b, rhIFNα2b) has been carried out. The V, D and J-gene segments, the complementaritydetermining (CDR) and framework (FR) regions, n-nucleotides as well as mutation...
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Інститут клітинної біології та генетичної інженерії НАН України
2009
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irk-123456789-666232014-07-20T03:01:48Z In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b Flyak, A.I. Pavlova, M.V. Gilchuk, P.V. Оригинальные работы In silico analysis of the DNA encoding singlechain Fv antibodies (ScFv) specific to the human recombinant interferon β1b and α2b (rhIFNβ1b, rhIFNα2b) has been carried out. The V, D and J-gene segments, the complementaritydetermining (CDR) and framework (FR) regions, n-nucleotides as well as mutation rates which take place during the affinity maturation of the examined sequences have been determined. For the panel of ScFv against rhIFNβ1b isolated from an immune combinatorial cDNA library uniqueness of the CDRH3 loop by the length and amino acid composition has been shown. Multiple alignments with the nearest homologies from the NCBI databases have revealed that the sequences of ScFv obtained are new. Проведен in silico анализ структуры последовательностей ДНК, кодирующих специфические к интерферону β1b и α2b человека (rhIFN β1b, rhIFN α2b) одноцепочечные антитела (ScFv – single chain Fv): определены V, D и J сегменты, границы антиген связывающих (CDR) и каркасных (FR) участков, n нуклеотиды, а также величина мутационных процессов, которые имели место при аффинном дозревании последовательностей in vivo. Для представителей панели ScFv против rhIFN β1b, изолированных из иммунной комбинаторной библиотеки кДНК V генов, показана уникальность участка CDRH3 как по длине, так и по аминокислотному составу. Множественное выравнивание с ближайшими гомологами базы данных NCBI показало, что полученные нами последовательности ScFv являются новыми. Проведено in silico аналіз структури послідовностей ДНК, які кодують специфічні до рекомбінантного інтерферону β1b та α2b людини (rhIFN β1b, rhIFN-α2b) одноланцюгові антитіла (ScFv – single chain Fv): визначено V , D та J генні сегменти, межі антигензв’язувальних (CDR) та каркасних (FR) ділянок, n- нуклеотиди, а також величину мутаційних процесів, що мали місце при афінному дозріванні даних послідовностей in vivo. Для представників панелі ScFv проти rhIFN β1b, ізольованих з імунної комбінаторної бібліотеки кДНК V генів, показано унікальність ділянки CDRH3 як за довжиною, так і за амінокислотним складом. Множинне вирівнювання з найближчими гомологами бази даних NCBI показало, що одержані нами послідовності ScFv є новими. 2009 Article In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b / A.I. Flyak, M.V. Pavlova, P.V. Gilchuk // Цитология и генетика. — 2009. — Т. 43, № 1. — С. 54-60. — Бібліогр.: 19 назв. — англ. 0564-3783 http://dspace.nbuv.gov.ua/handle/123456789/66623 577.27 en Цитология и генетика Інститут клітинної біології та генетичної інженерії НАН України |
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Оригинальные работы Оригинальные работы |
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Оригинальные работы Оригинальные работы Flyak, A.I. Pavlova, M.V. Gilchuk, P.V. In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b Цитология и генетика |
| description |
In silico analysis of the DNA encoding singlechain Fv antibodies (ScFv) specific to the human recombinant interferon β1b and α2b (rhIFNβ1b, rhIFNα2b) has been carried out. The V, D and J-gene segments, the complementaritydetermining (CDR) and framework (FR) regions, n-nucleotides as well as mutation rates which take place during the affinity maturation of the examined sequences have been determined. For the panel of ScFv against rhIFNβ1b isolated from an immune combinatorial cDNA library uniqueness of the CDRH3 loop by the length and amino acid composition has been shown. Multiple alignments with the nearest homologies from the NCBI databases have revealed that the sequences of ScFv obtained are new. |
| format |
Article |
| author |
Flyak, A.I. Pavlova, M.V. Gilchuk, P.V. |
| author_facet |
Flyak, A.I. Pavlova, M.V. Gilchuk, P.V. |
| author_sort |
Flyak, A.I. |
| title |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| title_short |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| title_full |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| title_fullStr |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| title_full_unstemmed |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| title_sort |
in silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b |
| publisher |
Інститут клітинної біології та генетичної інженерії НАН України |
| publishDate |
2009 |
| topic_facet |
Оригинальные работы |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/66623 |
| citation_txt |
In silico analysis of the structure of variable domains of mouse single-chain antibodies specific to the human recombinant interferon β1b / A.I. Flyak, M.V. Pavlova, P.V. Gilchuk // Цитология и генетика. — 2009. — Т. 43, № 1. — С. 54-60. — Бібліогр.: 19 назв. — англ. |
| series |
Цитология и генетика |
| work_keys_str_mv |
AT flyakai insilicoanalysisofthestructureofvariabledomainsofmousesinglechainantibodiesspecifictothehumanrecombinantinterferonb1b AT pavlovamv insilicoanalysisofthestructureofvariabledomainsofmousesinglechainantibodiesspecifictothehumanrecombinantinterferonb1b AT gilchukpv insilicoanalysisofthestructureofvariabledomainsofmousesinglechainantibodiesspecifictothehumanrecombinantinterferonb1b |
| first_indexed |
2025-07-05T16:49:58Z |
| last_indexed |
2025-07-05T16:49:58Z |
| _version_ |
1836826439447478272 |
| fulltext |
УДК 577.27
A.I. FLYAK 1, M.V. PAVLOVA 1, P.V. GILCHUK 2
1 National Taras Shevchenko University of Kyiv, Ukraine
2 Institute of Molecular Biology and Genetics NAS of Ukraine, Kyiv
E�mail: gilchuk@ukr.net
E�mail: flyaka@mail.ru
IN SILICO ANALYSIS OF THE
STRUCTURE OF VARIABLE DOMAINS
OF MOUSE SINGLE�CHAIN
ANTIBODIES SPECIFIC TO THE HUMAN
RECOMBINANT INTERFERON β1b
In silico analysis of the DNA encoding single�chain Fv
antibodies (ScFv) specific to the human recombinant interfer�
on β1b and α2b (rhIFN�β1b, rhIFN�α2b) has been carried
out. The V�, D� and J�gene segments, the complementarity�
determining (CDR) and framework (FR) regions, n�nucleotides
as well as mutation rates which take place during the affinity
maturation of the examined sequences have been determined.
For the panel of ScFv against rhIFN�β1b isolated from an
immune combinatorial cDNA library uniqueness of the
CDRH3 loop by the length and amino acid composition has
been shown. Multiple alignments with the nearest homologies
from the NCBI databases have revealed that the sequences of
ScFv obtained are new.
Introduction. An important feature of antibodies
is the extraordinary variability of their antigen�bind�
ing sites ensuring specific and high�affinity interac�
tion with the target antigen. Due to their high bind�
ing ability antibodies are of great value as unique
molecular probes for fundamental research, as well
as in biotechnology and medicine.
Modern gene manipulation techniques enable
generation of recombinant antibody fragments –
single�chain antibodies (ScFv’s), as well as their
production in Escherichia coli cells. ScFv’s are
obtained by translating DNA sequences of immu�
noglobulin heavy�chain (VH) and light�chain (VL)
variable domains joined in one gene [1]. The appro�
aches to construction and selection of combinato�
rial cDNA libraries of V�genes in vitro allow isola�
tion of ScFv with desired affinity and specificity,
bypassing the stages of traditional hybridoma tech�
nology [2, 3].
The diversity of antigen�binding regions of anti�
body variable domains is defined by the structure
of the underlying encoding gene elements. The
genes of the light chain result from recombination
of one variable (V) and joining (J) gene segments.
Recombination events in the heavy chain are more
complex due to the presence of an additional D�
segment. One of the mechanisms of increasing diver�
sity is incorporation of n�nucleotides (nontem�
plated nucleotides) at the gene segment joint site
[4]. Later on, the DNA sequences of variable (V)
domains are affected by the influence of somatic
hypermutations taking place during the affinity
maturation of B�cells. The highest rate of point
mutations occurs at complementarity determining
regions (CDR), as opposed to framework regions
(FR) where the rate is relatively low.
Up to date, there have been created a number of
databases of antibody germline gene segments, as
well as software for identification of V�, D�, and J�
gene segments, n�nucleotides, CDR and FR seg�
ments, mutation rates, etc. A detailed analysis of
nucleotide and aminoacid sequences of antibody
V�domains is needed for a number of reasons. First
of all, it allows determining the degree of confor�
mational uniqueness of the antigen binding site of
the obtained antibodies, which can be achieved by
comparing their aminoacid sequences with yet
known immunoglobulin sequences. Second, while
analyzing multiple antibody variants against one
antigen (a panel of antibodies), determining V�, D�,
and J�gene segments, n�nucleotides, and mutations
rates enables evaluation of the panel’s heterogene�
ISSN 0564–3783. Цитология и генетика. 2009. № 154
© A.I. FLYAK, M.V. PAVLOVA, P.V. GILCHUK, 2009
ity and proper choice of the most promising candi�
date molecules for further work. However, what
makes most sense is that determining CDR and
FR regions of antibody V�domains is an indispen�
sable prerequisite for beginning manipulations to
improve their functional characteristics, such as
decreasing immunogeneity (in case of humaniza�
tion), or DNA combinatorial mutagenesis – an in
vitro technique used to increase affinity and stabil�
ity of obtained antibodies.
The most widespread strategy for humanization
is grafting of CDR regions of an antigen�specific
monoclonal antibody with FR regions of selected
V�domains of human antibodies [5]. An example
of the practical value of the abovementioned tech�
nology is the fact that today out of 30 monoclonal
antibody�based drugs approved by FDA (Food and
Drug Administration, USA) for clinical applica�
tion over one third are humanized antibodies [6].
The core of the combinatorial mutagenesis tech�
nique lies in directed randomization of certain
regions of V�domains using a set of oligonucleotides
with degenerate codons. Randomization targets may
include CDR3 regions of VH and VL domains [7],
all six CDRs [8], or certain aminoacids of V�
domains [9].
The indicated method is particularly valuable
while working with naїve and synthetic libraries of
antibody fragments («single�pot» antibody libraries),
as combined with the phage display it allows isola�
tion of antibody molecule variants with improved
characteristics of expression in bacteria, prolonged
stability in blood stream, as well as exclusively high
affinity to the target antigen [8].
Our previous work resulted in obtaining and
characterization of a panel of mouse ScFv’s against
rhIFN�β1b. The goal of the present work was se�
quencing and in silico analysis of the primary
structure of the ScFv’s obtained.
Materials and methods. DNA sequencing was
carried out using the automatic DNA sequencer
IBI Prism 3130 (Applied Biosystems, USA). We
sequenced DNA (approx. 1000 bp) obtained by
polymerase chain reaction (PCR) using the follow�
ing primers: pCANTAB�R1 5'�d [CCATGATTA�
CGCCAAGCTTTGGAGCC]�3', pCANTAB�R2
5'�d [CGATCTAAAGTTTTGTCGTCTTTCC]�3'
and preliminary obtained recombinant phagemids
of the corresponding bacterial clones as matrices.
For sequencing, we used the following primers:
pCANTAB 5�S3: 5'�d [GGTTCAGGCGGAGGTG�
GCTCTGG] – 3';
pCANTAB 5�S4: 5'�d [CCAGAGCCACCTCCGCC�
TAACC] – 3',
pCANTAB 5�S1: 5'�d [CAACGTGAAAAAATTAT�
TATTCGC] – 3';
pCANTAB 5�S6: 5'�d [GTAAATGAATTTTCTGTAT�
GAGG] – 3'.
Sequencing results were processed and interpret�
ed with assistance of Vector NTI Advance 10 (Invi�
trogen, USA) software. The level of homology bet�
ween ScFv nucleotide sequences was determined
using ClustalW2 software (http://www.ebi.ac.uk /
Tools/clustalw2/) [10]. Identification of V�, D�, and
J�gene segments encoding ScFv specific to rhIFN�
β1b and rhIFN�α2b, was assisted by Somatic
Diversification Analysis (SoDA) software (http://
dulci.org/soda/) [11]. All gene segment names are
used in accordance with the standardization rules of
the international information system IMGT [12].
Additional n�nucleotides and mutations that oc�
curred during antibody affinity maturation were
determined by comparing the target sequences
with respective germline gene sequences of V�, D�,
and J�gene segments from the IMGT database.
CDR and FR regions were determined with the
help of SoDA software in accordance with the crite�
ria developed by IMGT. In view of this, we labeled
the indicated regions as CDR�IMGT and FR�
IMGT [13].
Search of closest homologs for the aminoacid
sequences was performed using IgBLAST software
(http://www.ncbi.nlm.nih.gov/igblast/) of the im�
munoglobulin V region sequences database of the
National Center for Biotechnology Information
(NCBI), USA. First, we looked for homologs to a
whole ScFv sequence, and then separately for vari�
able domains VH and VL. For sequences with high�
est homology, we built multiple alignments using
Vector NTI Advance 10 software.
Results and Discussion. In previous publica�
tions, we described generation of an immune com�
binatorial cDNA library of mouse immunoglobu�
lin V�genes and isolation of a panel of single�chain
antibodies specific to rhIFN�β1b [14, 15]. For
some isolated ScFv’s such characteristics as bind�
ing specificity to native and denatured antigen,
level of production in E. coli by secretion, stability
etc. were determined. An interesting point was that
some producers randomly selected out of more
ІSSN 0564–3783. Цитология и генетика. 2009. № 1 55
In silico analysis of the structure of variable domains of mouse single�chain
than 30 isolated positive clones invariably differed
between each other in the production level of the
ScFv’s (0.6 to 3.5 mg/L) and their accumulation
rate, ScFv’s sensitivity to proteolysis, ScFv’s affini�
ty (1.96 · 10–8 M to 1.69 · 10–9 M), and ScFv’s bind�
ing specificity with native and denatured rhIFN�
β1b [15].
In many experiments dedicated to study expres�
sion of ScFv in E. coli, the influence of the primary
structure of variable domains on their production
rate has been shown. It is also known that replace�
ment of at least one aminoacid in CDR regions
may have dramatic influence on antibody affinity
and specificity [16]. The differences in production
rates in bacterial cells, as well as various antigen�
binding characteristics of some ScFv’s from the
obtained panel seem to suggest nonidentity of the
primary structure of their variable domains. To test
this suggestion we sequenced the DNA of all
ScFv’s identified as positive in the reaction of
binding to rhIFN�β1b. Among the 12 analyzed
sequences isolated from different producers, we
revealed four DNA variants that encode ScFv with
different primary structure – #1, #2, #4, #11.
These were the sequences we selected for further
analysis.
DNA homology of ScFv’s was estimated using
ClustalW2 software [10]. For analysis, we used the
four sequences of ScFv’s against rhIFN�β1b iso�
lated from the immune library, a sequence of ScFv
against rhIFN�β1b preliminarily obtained from a
naїve library [15], as well as a sequence of ScFv’s
against rhIFN�α2b [17]. According to the criteri�
on of homology for their DNA, all six ScFv’s were
provisionally divided into four groups (Figure).
Mapping structurally and functionally valuable
regions within antibody variable domains is an
important task. Using SoDA software, we identi�
fied V�, D�, and J�gene segments, n�nucleotides,
as well as mutations that occurred during affinity
maturation of respective sequences in vivo [11].
Armed with this same software we identified the
boundaries and lengths of all CDR�IMGT and
FR�IMGT. The results are summarized in Table 1.
It was determined that the DNA sequences of
VH and VL domains of the ScFv’s from the first
group (#1, #4) were formed by germline gene seg�
ments IGHV1–26 and IGKV3–2; the sequences
ISSN 0564–3783. Цитология и генетика. 2009. № 156
A.I. Flyak, M.V. Pavlova, P.V. Gilchuk
A dendrograme reflecting the homology between nucleotide
sequences of ScFv’s carried out using ClustalW2 software.
#1, #2, #4, #11 are ScFv’s isolated from an immune
cDNA library and specific to rhIFN�β1b; ScFv (naЇve) –
ScFv’s isolated from a naїve cDNA library and specific to
rhIFN�β1b; ScFv (IFNα) – ScFv’s specific to rhIFN�α2b
Table 1
ScFv’s variable domain DNA analysis carried out using SoDA software
Group ScFv
V�
domain
V�gene
segment
Number
of V�
mutations
Number
of V�D n�
nucleotides
(V�J for VL)
D�gene
segment
Number
of D�
mutations
Number
of D�J n�
nucleotides
J�gene
segment
Number of
J�muta�
tions
CDRH3
region
length
1
2
3
4
1
4
2
11
ScFv
(naive)
ScFv
(IFNα)
H
L
H
L
H
L
H
L
H
L
H
L
IGHV1�26
IGKV3�2
IGHV1�26
IGKV3�2
IGHV1�67
IGKV3�2
IGHV1�67
IGKV3�2
IGHV1S128
IGKV17�121
IGHV14�3
IGKV8�19
23
5
24
9
15
8
18
5
10
0
6
11
3
0
3
0
2
4
4
4
2
2
3
4
IGHD4�1
IGHD4�1
IGHD4�1
IGHD3�2
IGHD2�1
IGHD1�1
0
0
0
1
1
0
2
4
3
0
2
0
IGHJ2
IGKJ1
IGHJ1
IGKJ1
IGHJ4
IGKJ5
IGHJ1
IGKJ5
IGHJ2
IGKJ1
IGHJ4
IGKJ1
3
4
2
2
4
2
2
2
4
2
4
2
6
9
6
9
6
9
6
9
13
9
10
9
Note. All V�, D�, and J�gene segments’ names are in accordance with the IMGT standardization rules [12].
of the second group (#2, #11) were formed by the
segments IGHV1–67 and IGKV3–2; those of the
third group – by IGHV1S128 and IGKV17–121;
and those of the fourth group – by IGHV14–3 and
IGKV8–19, respectively. The results suggest that
all the V�gene segment of the four ScFv’s from the
panel isolated from the immune combinatorial
cDNA library belong to the first and the third
IMGT subgroup, respectively [12]. The length of
CDRH3�IMGT and CDRL3�IMGT regions of
the four ScFv’s isolated from the immune library
was 6 and 9 aminoacid residues, respectively. The
CDRH3�IMGT region of ScFv isolated from the
naїve library turned out to be longer and consisted
of 13 aminoacid residues (Table 1).
One of V�genes’ diversity sources are somatic
hypermutations appearing in vivo during the process�
es known as B�cell affinity maturation. Random
ІSSN 0564–3783. Цитология и генетика. 2009. № 1 57
In silico analysis of the structure of variable domains of mouse single�chain
Table 2
Alignment of CDR�IMGT regions for VH and VL of obtained ScFv’s with nearest V�domain sequences 1
Group № of sequence CDR1 CDR2 CDR3
Regions for VH
Regions for VL
1
2
3
4
1
4
AAO852802
AAO852842
AAO852862
ABK599112
2
11
AAF814172
CAA749172
CAA749232
AAA832672
ScFv(naЇve)
AAA832672
ScFv(IFNa)
AAA165832
GYSFTGYP
********
*******T
*******T
*******T
*******T
GYTFTDYA
********
********
**K*****
**K*****
*****S*W
GYTFTSYR
*******W
GFNIKDTF
*******Y
INPYNGGT
********
********
********
********
********
ISTYSGDT
******N*
****N*N*
****Y***
****Y***
*NPSN*R*
INPSNGRT
********
IDPANGYT
******N*
ARF---------PA--Y
***---------**--*
**YPQFITTARRY*MD*
**AAG---ILRLRDFD*
**AAG---ILRLRDFD*
**DVRG------AWFA*
ARP---------PN--Y
***---------*A--*
**YYG-------NYFD*
*LLRP---------FA*
*LLRP---------FA*
**GGVYY-DLYYYALDY
ARSYYG----NFYYFDY
**GGVYY-DLYY*AL**
ASR-------VDYAMDY
*R*-------AS*****
1
2
3
4
1
4
ABC553232
1H0D_A2
AAA390152
S099692
2
11
P016562
AAB304602
CAA801072
P016542
ScFv(naЇve)
AAZ503752
ScFv(IFNa)
AAA387302
ESVDNYGIS--F
*********--*
*********--*
*********--*
*********--*
*********--*
ESVDKYGIS--F
****N****--*
****NS***--*
****N****--*
****N****--*
****N****--*
TDIDDD------
******------
QSLLNSGNQKNY
************
AAS
***
***
***
***
***
AAS
***
***
***
***
***
EGN
***
WAS
***
QQSKEVPWT
**G******
*******Y*
*******L*
*********
**G**I*Y*
QQSKEVPWT
*********
*********
*********
*******P*
*********
LQSDNLPLP
********T
QNDYSYPLT
*********
Note. 1 – search of closest homologs for the aminoacid sequences was performed using IgBLAST software of the
immunoglobulin V�region sequences database of the NCBI; 2 – the sequence number corresponds to the number in
GenBank; «_» indicated amino acids common for CDR�IMGT regions of ScFv’s of groups 1 and 2; * indicated the same
amino acids within each ScFv group; «�» – denotes the absence of an aminoacid at this position.
aminoacid replacements affect the antigen�binding
properties of antibodies, which results from local
changes in variable domain conformation as well
as changes in quantity/quality of functional groups
in aminoacid residues that directly contact with
antigen [8]. For V�, D�, and J�gene segments of V�
domains of the studied ScFv’s, we have determined
the respective mutation rates – the absolute quan�
tity of nucleotide substitutions deduced from juxta�
posing the sequences in question against the
sequences of the identified germline gene segments
(Table 1). It has been shown that the indicated rates
for V�gene segments of ScFv’s #1 and #4 (group 1)
is higher compared to that of ScFv #2 and #11
(group 2). The fact that the absolute mutation rate
for V�gene segments of ScFv isolated from the naїve
library (ScFv (naїve)) is the lowest for all analyzed
sequences was an expected result (Table 1).
Our search results for similar experimental works
suggest that a panel of ScFv’s against rhIFN�β1b
has been obtained for the first time. Taking into
account this fact, as well as the methodological
novelty of the generation and selection scheme for
the respective combinatorial library of V�genes [14],
it was important to estimate the degree of unique�
ness of the antigen�binding site of the isolated
ScFv’s. It is common knowledge that the specifici�
ty of the «antigen�antibody» interaction is, first of
all, determined by the primary structure of the
CDR regions which aminoacids are responsible
for direct contact with the antigen. Exposed on the
surface of V�domains, CDR regions are spatially
accessible for interaction with the antigen and
form canonical loop�like structures. Among other
CDRs, CDRH3 is the most variable in length and
aminoacid composition, and plays a key role in
antigen recognition [16].
Using IgBLAST software of the NCBI immuno�
logic sequence database we performed a search for
closest homologs separately for the aminoacid
sequences of both V�domains and the whole
sequence of ScFv. Multiple alignment of the
obtained ScFv’s with their closest homologs from
the database revealed differences in the primary
structures of VH and VL in aminoacid substitutions
at certain positions (results not presented), while
the structure of CDRH3�IMGT turned out to be
absolutely unique (Table 2). Uniqueness of the pri�
mary structure of CDRH3�IMGT as well as the
absence of antibody sequences with homologous
V�domains in the database, suggests that the anti�
gen�binding site of the four isolated ScFv is con�
formationally unique, and the sequences are novel.
Comparative analysis of the CDRH3�IMGT
regions of these ScFv’s has revealed a conservative
motif AR_P_Y with substitutions at positions 120
(F/P) and 122 (A/N). Substantial differences were
observed in CDRH1�IMGT and CDRH2�IMGT
(Table 2). It is known from published data that
only a slight part of the aminoacid residues of
CDRs contribute significantly to the free energy
(�G) of interaction with the antigen [18]. Some
publications show a key role for aminoacids located
in the central part of CDRH3, while the flanking
aminoacids mostly stabilize the formed complex
[8, 16, 19]. It is a matter of fact that single aminoacid
substitutions in the central part of CDRH3 may
dramatically affect antibody’s affinity and speci�
ficity. The aforementioned substitutions in CDR�
IMGT may explain the differences in the panel’s
ScFv’s antigen�binding properties studied in the
previous work [15]. We suppose the identified
amino acid residues are promising targets for fur�
ther ScFv’s mutagenesis.
It seemed interesting to compare the structure
of CDRH3�IMGT regions of ScFv’s specific to
antigens rhIFN�β1b and rhIFN�α2b that are
structural and functional homologs. For compari�
son, we used the sequences of two highly specific
ScFv’s isolated from different independently gen�
erated immune libraries. For all six CDR�IMGT
of ScFv’s that were compared, we observed differ�
ences in the amino acid composition to the extant
of individual substitutions, while CDRL1�IMGT
and CDRH3�IMGT also differed by the length of
the loop (Table 2, sequences #1 and ScFv (IFNα)).
Taking into account the absence of crosslink bind�
ing between these ScFv’s and the respective antigens,
which we had shown before using ELISA, we may
suppose their interaction with different epitopes.
Conclusion. Within a panel of ScFv’s against
rhIFN�β1b isolated from an immune combinator�
ial cDNA library of mouse genes, we have revealed
four sequences with different primary structures.
In silico analysis has identified structurally and
functionally important regions in the variable
domains of the obtained ScFv’s. It has been shown
that the structure of the antigen�binding site of the
four isolated ScFv’s is unique, and the sequences
themselves are new.
ISSN 0564–3783. Цитология и генетика. 2009. № 158
A.I. Flyak, M.V. Pavlova, P.V. Gilchuk
Authors are grateful to staff of Department of
Functional Genomics of Institute of Molecular
Biology and Genetics NAS of Ukraine for DNA
sequencing.
А.И. Фляк, М.В. Павлова, П.В. Гильчук
IN SILICO АНАЛИЗ СТРУКТУРЫ
ВАРИАБЕЛЬНЫХ ДОМЕНОВ
ОДНОЦЕПОЧЕЧНЫХ АНТИТЕЛ МЫШИ,
СПЕЦИФИЧНЫХ К РЕКОМБИНАНТНОМУ
ИНТЕРФЕРОНУ β1b ЧЕЛОВЕКА
Проведен in silico анализ структуры последователь�
ностей ДНК, кодирующих специфические к интерфе�
рону β1b и α2b человека (rhIFN�β1b, rhIFN�α2b) од�
ноцепочечные антитела (ScFv – single�chain Fv): опре�
делены V, D и J сегменты, границы антиген�связыва�
ющих (CDR) и каркасных (FR) участков, n�нуклеоти�
ды, а также величина мутационных процессов, кото�
рые имели место при аффинном дозревании последо�
вательностей in vivo. Для представителей панели ScFv
против rhIFN�β1b, изолированных из иммунной ком�
бинаторной библиотеки кДНК V�генов, показана
уникальность участка CDRH3 как по длине, так
и по аминокислотному составу. Множественное вы�
равнивание с ближайшими гомологами базы данных
NCBI показало, что полученные нами последователь�
ности ScFv являются новыми.
А.І. Фляк, М.В. Павлова, П.В. Гільчук
IN SILICO АНАЛІЗ СТРУКТУРИ
ВАРІАБЕЛЬНИХ ДОМЕНІВ
ОДНОЛАНЦЮГОВИХ АНТИТІЛ МИШІ,
СПЕЦИФІЧНИХ ДО РЕКОМБІНАНТНОГО
ІНТЕРФЕРОНУ β1b ЛЮДИНИ
Проведено in silico аналіз структури послідовнос�
тей ДНК, які кодують специфічні до рекомбінантного
інтерферону β1b та α2b людини (rhIFN�β1b, rhIFN�
α2b) одноланцюгові антитіла (ScFv – single�chain Fv):
визначено V�, D� та J�генні сегменти, межі антиген�
зв’язувальних (CDR) та каркасних (FR) ділянок, n�
нуклеотиди, а також величину мутаційних процесів,
що мали місце при афінному дозріванні даних послі�
довностей in vivo. Для представників панелі ScFv про�
ти rhIFN�β1b, ізольованих з імунної комбінаторної бі�
бліотеки кДНК V�генів, показано унікальність ділян�
ки CDRH3 як за довжиною, так і за амінокислотним
складом. Множинне вирівнювання з найближчими
гомологами бази даних NCBI показало, що одержані
нами послідовності ScFv є новими.
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