Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination
Aim: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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irk-123456789-1391032018-06-20T03:10:15Z Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination Meleshko, A.N. Vashkevich, K.P. Fomina, E.G. Scheslenok, E.P. Schkolina, T.V. Sergeev, G.V. Original contributions Aim: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. Methods: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. Results: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. Conclusion: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6–7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration. 2013 Article Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination / A.N. Meleshko, K.P. Vashkevich, E.G. Fomina, E.P. Scheslenok, T.V. Schkolina, G.V. Sergeev // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 8-14. — Бібліогр.: 34 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139103 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Meleshko, A.N. Vashkevich, K.P. Fomina, E.G. Scheslenok, E.P. Schkolina, T.V. Sergeev, G.V. Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination Experimental Oncology |
| description |
Aim: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and expression of recombinant idiotype protein in Escherichia coli. Methods: PCR with specially designed panel of primers was used for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. Results: Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. Conclusion: The proposed method of vaccine production is relatively cheap, not very laborious and requires as long as 6–7 week to perform. The expressed protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration. |
| format |
Article |
| author |
Meleshko, A.N. Vashkevich, K.P. Fomina, E.G. Scheslenok, E.P. Schkolina, T.V. Sergeev, G.V. |
| author_facet |
Meleshko, A.N. Vashkevich, K.P. Fomina, E.G. Scheslenok, E.P. Schkolina, T.V. Sergeev, G.V. |
| author_sort |
Meleshko, A.N. |
| title |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination |
| title_short |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination |
| title_full |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination |
| title_fullStr |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination |
| title_full_unstemmed |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination |
| title_sort |
cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human scfv protein in e. coli for anti-idiotype vaccination |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2013 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/139103 |
| citation_txt |
Cloning of variable fragments of tumor immunoglobulin, assembling and expressing of human SCFV protein in E. coli for anti-idiotype vaccination / A.N. Meleshko, K.P. Vashkevich, E.G. Fomina, E.P. Scheslenok, T.V. Schkolina, G.V. Sergeev // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 8-14. — Бібліогр.: 34 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T07:36:25Z |
| last_indexed |
2025-07-10T07:36:25Z |
| _version_ |
1837244603585003520 |
| fulltext |
8 Experimental Oncology 35, 8–14, 2013 (March)
CLONING OF VARIABLE FRAGMENTS OF TUMOR
IMMUNOGLOBULIN, ASSEMBLING AND EXPRESSING OF HUMAN
SCFV PROTEIN IN E. COLI FOR ANTI-IDIOTYPE VACCINATION
A.N. Meleshko1,*, K.P. Vashkevich1, E.G. Fomina2, E.P. Scheslenok2, T.V. Schkolina2, G.V. Sergeev3
1Belarusian Research Center for Pediatric Oncology, Hematology and Immunology, Minsk 223053, Belarus
2Republican Research and Practical Center for Epidemiology and Microbiology, Minsk 220114, Belarus
3Institute of Bioorganic Chemistry, Minsk 220141, Belarus
Aim: Idiotype, the unique part of immunoglobulin molecule expressed on the surface of B-cells, represents a specific antigen for
vaccination against lymphoma. We have developed a rapid method for immunoglobulin variable fragments cloning, assembling and
expression of recombinant idiotype protein in Escherichia coli. Methods: PCR with specially designed panel of primers was used
for direct amplification of variable regions of tumor immunoglobulin. Overlapping extension PCR, restriction and ligation was
applied for assembling and cloning of vaccine construction. Idiotype protein was purified by metal-chelate chromatography. Results:
Methods of idiotype cloning from lymphoma cells and production of recombinant protein were developed and optimized. Several
samples of idiotypic proteins originating from B-cell lines and lymphoma patients were produced. Conclusion: The proposed
method of vaccine production is relatively cheap, not very laborious and requires as long as 6–7 week to perform. The expressed
protein was soluble, did not accumulate in inclusion bodies and harvested at sufficient for vaccination quantity and concentration.
Key Words: immunoglobulin, idiotype, vaccine, lymphoma.
Development of anti-cancer vaccination has still
been limited by the low number of known tumor-
associated antigens that might be used as targets for
directional immunization. Cells of lymphomas express
antigen-specific receptors, surface immunoglobulin
for B cell lymphomas and T cell receptor for lympho-
mas of the T lineage. As tumor has a clonal origin, all
cells of lymphoma carry identical immunoglobulin
or TCR receptor on their surface. Therefore, variable
domains of immunoglobulin (idiotype) can be used
as specific antigen for anti-idiotypic vaccination.
The clinical studies of anti-idiotypic vaccination have
started since 1992 when Ron Levy and Larry Kwak (Stan-
ford University, USA) first vaccinated follicular lymphoma
patients with tumor-derived immunoglobulin [1]. In the
last decade numerous clinical trials were performed
using hybridoma-prodused immunoglobulin harvested
from cell supernatant and showed immunological re-
sponse and clinical benefit in vaccinated patients [2, 3].
While preparation of individual Ig using rescue hy-
bridoma method is time-consuming and expensive,
further studies came to production of recombinant vac-
cine by cloning variable Ig fragments from DNA of tumor
cells [4]. Several methods for expression of Id-protein
have been used, including insect cell culture [5], plants
[6–8], bacteria E. coli [9, 10], or cell-free expression
system [11, 12]. These methods require some efforts
for purification of protein. Id-protein can be conjugated
with immunogenic carrier protein, such as KLH, and ad-
ministrated to the patient with adjuvant, usually GM-CSF
[13]. Alternatively, production of Id-pulsed autologous
dendritic cells (DC) can be used for enhancing anti-
tumor immune response [14, 2]. Finally, the simplest
strategy is nude DNA vaccines [15–17].
Most methods of recombinant idiotype protein
expression and all methods of DNA Id-vaccine utilize
cloning of heavy and light immunoglobulin chains vari-
able fragments and their assembling into single chain
variable fragment (scFv) [18]. The most difficult problem
in that procedure is PCR amplification of the whole vari-
able regions of both immunoglobulin chains due to great
number of VH, VK and VL gene segments and their oc-
casional usage in Ig genes rearrangement. To solve this
problem, a sophisticated method of anchored-PCR for
amplification 5’-end of cDNA was developed [19]. This
method implies addition of oligo-dG-tail to the 5’-end
of cDNA by enzyme Tdt, first amplification with anchor-
primer and subsequent semi-nested amplification
with internal primer to the proximal constant region.
Anchored-PCR requires optimization to be performed
properly. In original publication of F. Osterroth only
amplification of IgM heavy chain and IgK light chain
was described [19]. Several studies describe amplifica-
tion of V-regions with specific primers and assembling
of scFv [20, 21] for defined immunoglobulin or hybri-
doma, which is not suitable for broad applications.
In this study, we performed design of specific
primers for direct amplification of all possible variable
regions of IgM, IgG heavy chains, IgK, IgL light chains
without loss or addition of amino acids, and describe
method of cloning, assembling scFv and expression
of idiotypic protein in bacteria E. coli. As well, we re-
alized the ability to add fusion gene of co-stimulator
to the construction in the N-part of the protein. Recom-
binant Id-protein obtained in this way may be used for
direct vaccination or loading of DC.
MATERIALS AND METHODS
Cell lines, patient samples, RNA isolation and
cDNA synthesis. Four immunoglobulin expressing
lymphoma cell lines IM-9, Daudi, RPMI1788 and Na-
Received: September 15, 2012.
*Correspondence: E-mail: meleshko@tut.by
Exp Oncol 2013
35, 1, 8–14
Experimental Oncology 35, 8–14, 2013 (March) 9
malva were used for idiotype cloning. Cells were grown
in RPMI-1640 medium with 10% FBS and 2 mM gluta-
mine. 1–5 × 106 cells were collected, washed with PBS
and lysed for RNA extraction. Tumor cells from patients
were obtained from bone marrow, lymph node biopsy
or ascitic fluid containing more than 80% of malignant
lymphoma cells by histological and/or immunopheno-
typing analysis. Material from three pediatric patients
was used in this study, ascitic fluid from patient 1 with
Burkitt’s lymphoma, tumor lymph node biopsy from
patient 2 with diffuse large B-cell lymphoma and tumor
lymph node biopsy from patient 3 with Burkitt’s lym-
phoma. Tissue was disrupted in 2 mL of saline solution
with 50 μL of RNAlater (Qiagen) to generate cell suspen-
sion. RNA was isolated TRI reagent (Sigma) according
to the manufacture’s instruction dissolved in sterile water,
measured by spectrophotometry and immediately stored
at −80°C. 1 μg of total RNA was used for cDNA synthesis
with MMLV reverse transcriptase and Oligo-dT.
PCR amplification of Ig variable fragments. PCR
amplification was performed for each gene in a semi-
nested manner with a specially designed primers panel
(details in Results). All primers were designed to unify
PCR conditions at 60 °C. PCR reaction was performed
in 30 μL with 12.5 pmol of each primer, 200 μM dNTP,
1.5 mM MgCl2 and 1U HF DNA-Polymerase (mix of Pfu
and Taq-polymerases, Primetech, Belarus). We used
30 cycles of PCR, but the number of cycles may be re-
duced to 20 cycles for the first step of amplification
to minimize probability of nucleotide replacements
due to DNA-polymerase errors. PCR products were
examined by 1.5% agarose gel. For the first step of am-
plification heteroduplex analysis in polyacrylamide gel
was applied to discriminate homoduplexes (mono-
clonal PCR products) from a smear of slowly moving
heteroduplexes (derived from polyclonal lymphocytes).
This step is useful only for tumor biopsy from patients
and not essential for B-cell lines. PCR products were
denatured for 5 min at 95 °C and rapidly cooled down
to +4 °C to induce duplex formation. Size separation
of the generated homoduplexes and heteroduplexes
was obtained in 8% non-denaturing polyacrylamide gel.
Molecular cloning and sequencing. Assembled
scFv fragment was excised from agarose gel, purified
by QIAquick Gel Extraction Kit (Qiagen) and directly li-
gated into pTZ57R/T vector through T/A overlapping us-
ing InsTAclone PCR Cloning Kit (Fermentas). E. coli XL1-
blue was transformed by calcium chloride method with
ligation mix. Selection of blue and white recombinant
clones was performed on S-Gal®/LB Agar Blend (Sig-
ma-Aldrich) with ampicillin. Plasmid DNA was recovered
from 5–10 clones with EasyPrep Pro Plasmid Miniprep
Kit (PerkinElmer). Sequence of insertion was done with
plasmid primers pUC19-seq-F GTTTTCCCAGTCAC-
GACGTT and pUC19-seq-R TGTGGAATTGTGAGCGGA-
TA. Sequence reaction was done using BigDye® Termi-
nator v3.1 Cycle Sequencing Kit (Applied Biosystems)
on genetic analyzer ABI PRISM 3130. Plasmids with
correct sequence were digested with NdeI and HindIII,
SalI or NotI restrictases (Fermentas) and subcloned
into pET24b (Novagen) expression vector. Before final
transformation sequence was checked by sequencing
with primers pET24b_F TGTGAGCGGATAACAATTCC
and pET24b_R TTCCTTTCGGGCTTTGTTAG.
Expression and purification of protein. Re-
combinant expression vectors with inserted idiotype
pET24b-scFv were introduced into bacteria E. coli BL21-
CodonPlus (DE3)-RIPL and selected by chlorampheni-
col and kanamycin. Night culture was diluted 1:200 with
TB medium, containing 30 mkg/ml kanamycin and
cultured at 37 °C until culture reached OD A600 0.6–0.8.
Expression was induced by cultivation with 0.5 mM IPTG
at 31 °C for 3 hours. Cells were harvested, resuspended
in 20 mM Tris-НСI, 0.4 mM PMSF, 0.5 M NaCl (рН 8.0)
buffer and disrupted with EmulsiFlex-C5 homogenizer
or treated with ultrasound three times for 20 s on ice.
Cell debris were separated by 20 min centrifugation
18 500 g and supernatant collected for electrophoresis
and protein purification.
Purification of protein was performed by metal-
chelate chromatography on columns with Ni-NTA
agarose. Binding of protein on the columns, washing
and elution was performed in denaturing conditions
with 6M urea. Column was washed by 20 mM imidazole
solution and protein was eluted by 300 mM imidazole.
Urea, salts and imidazole were removed by dialysis
[22]. Cell lysate, fraction of column wash and pure
protein were analyzed in 12% SDS-PAGE (Laemmli
method) and stained with Coomassie Brilliant Blue.
Western blot analysis. Samples of 10 ng of puri-
fied protein were run in 12% SDS-PAGE with positive
and negative controls. Transfer was done on Semi-dry
transport unit (Amersham Biosciences) onto nitrocel-
lulose filter. Filter was pre-stained with reversible dye
Ponceau S (Sigma-Aldrich) to check protein bands.
Blot was blocked with Western Blocker™ Solution
(Sigma-Aldrich), washed with Tween-TBS and treated
with primary monoclonal mouse antibodies to 6-His tag
(Abcam). Secondary antibodies were rabbit polyclonal
to mouse IgG conjugated to HRP (Abcam). Finally blot
was stained with diaminobenzene and documented with
ChemiDoc™ XRS+ System (Bio-Rad) in visible light.
Defined 6His-tagged protein with molecular weight
of 17 kDa was used as a positive control. Lines of filter
with separated tested and control proteins were also
treated with normal mice serum as negative control.
RESULTS
Primers design for PCR-amplification of im-
munoglobulin variable regions. Most human B-cell
lymphoma express immunoglobulin of the IgM isotype,
with either kappa or lambda light chain. However, some
lymphomas express IgG immunoglobulin [23]. To cover
as many lymphomas as possible, we developed extend-
ed primer panel for all four immunoglobulin chain types:
IgM, IgG, IgK and IgL. Database of all functional V gene
segments was collected form http://www.imgt.org web-
site. Immunoglobulin heavy chain V-segments were
combined by homology into VH1–6 families and aligned
with AlignX service of Vector NTI 9.0 program package
10 Experimental Oncology 35, 8–14, 2013 (March)
(Invitrogen) and MEGA-4 software. Forward primer was
designed to the first 7–8 codons of a VH gene segment
avoiding variable and polymorphic nucleotide near the
3’end of the primer. Primer design allowed up to 2 mis-
matches with germline sequence. An example of primer
design for VH1 is shown on Fig. 1.
Fig. 1. Primer design for aligned gene segments of VH1 family.
Primer sequence is underlined and titled on the top. Variable
nucleotides are marked as bold blue letters
The same principle was used for forward primes
of all 6 VH families of IgH, all 3 Vk families of IgK and
1–5 VL families of IgL gene. For some IgL V-segments
three or four first codons were excluded from PCR
amplification to enable optimal primer design. Several
VL gene segments belonging to VL6,7,8,9 families
were not included due to their very rare occurrence
in immunoglobulin molecule.
Two reverse primers were selected for proximal (C1)
constant region of IgM, IgG, IgK genes for two steps
of semi-nested PCR amplification of variable Ig domains.
Distal reverse primer for the first step of PCR is denoted
as -3’, and proximal primer is denoted as -clon. For IgL
gene, one distal IGLC-3’ primer was selected for all IGLC
regions, but two different proximal primers were se-
lected, one for IGLC1 and another for IGLC2–7 constant
regions. Reverse primers to heavy chain include in the
cloned fragment a few amino acids (12 a.a. for IgM and
10 a.a. for IgG) on the 5’ edge of CH1 segments. This
part of constant region was left intentionally to retain with
6His-linker two-domain structure of scFv. The schematic
positions of all primers are shown on Fig. 2 and primers
sequences are shown in Table 1.
Detection of immunoglobulin expression on tu-
mor cells. Diagnostics of lymphoma includes immu-
nohpenotypic analysis by flow cytometry, including
surface immunoglobulin M or G and kappa/lambda light
chain detection. Patient’s 1 lymphoma cells expressed
IgM+/IgK+, patient’s 2 — IgG+/IgK+ and patient’s 3 —
IgM+/IgL+. We also performed immunophenotypic
analysis of all B-cell lines kept in our laboratory: REH
IgH-/IgK-, Raji IgM+/IgK-, IM-9 IgG+/IgK+, Daudi
IgM+/IgK+, RPMI1788 IgM+/IgL+, Namalva IgM+/IgL+.
Cell lines REH and Raji were excluded because they
do not express mature immunoglobulin on cell surface.
Other cell lines: IM-9, Daudi, RPMI1788 and Namalva
were used for immunoglobulin V-genes cloning. DNA
of all samples used in this study was analyzed by PCR
for clonal IgH gene rearrangements as described in our
previous publication [24]. This step is not required for
vaccine production, but may be useful for rapid identi-
fication or verification of V-region and junctional region.
Table 1. Sequences of primers for PCR-amplification of immunoglobulin
variable regions
Primer name Sequence Position
VH1-5’clon CAGGTGCAGCTGGTGCAGTCTGG forward
VH2-5’clon CAGGTCACCTTGAAGGAGTCTGG forward
VH3-5’clon GAGGTGCAGCTGGTGGAGTCTGG forward
VH4-5’clon CAGGTGCAGCTGCAGGAGTCGGG forward
VH5-5’clon GAGGTGCAGCTGGTGCAGTCTGG forward
VH6-5’clon CAGGTACAGCTGCAGCAGTCAGG forward
Cμ-3’ CTCTCAGGACTGATGGGAAGCC reverse distal
Cμ-clon GGAGACGAGGGGGAAAAG reverse proximal
IgG-3’ GCCTGAGTTCCACGACACC reverse distal
IgG-clon CAGGGGGAAGACCGATGG reverse proximal
Vκ1-5’clon GACATCCAGATGACCCAGTCTCC forward
Vκ2/3-5’clon GATATTGTGATGACCCAGACTCCA forward
IgκC-3’ CCCCTGTTGAAGCTCTTTGT reverse distal
IgκC-clon AGATGGCGGGAAGATGAAG reverse proximal
VL1_(51)_clon CAGTCTGTGTTGACGCAGCCGCCCTC forward
VL1_(36-47)_clon TCTGTGCTGACTCAGCCACCCTC forward
VL1_(40)_clon CAGTCTGTCGTGACGCAGCCGCCCTC forward
VL2-clon TCCGTGTCCGGGTCTCCTGGACAGTC forward
VL3-clon ACTCAGCCACCCTCGGTGTCAGTG forward
VL4-clon TCCTCTGCCTCTGCTTCCCTGGGA forward
VL5-clon CAGCCTGTGCTGACTCAGCC forward
IGLC-3’ GTGTGGCCTTGTTGGCTTG reverse distal
IGLC2-7_clon CGAGGGGGCAGCCTTGGG reverse proximal
IGLC1_clon AGTGACCGTGGGGTTGGCCTTGGG reverse proximal
Fig. 2. Scheme of primers positions and approximate length
of PCR-products
Experimental Oncology 35, 8–14, 2013 (March) 11
Amplification of immunoglobulin variable frag-
ments. PCR reactions were performed with primers
for identified immunoglobulin chain genes. Separate
PCR reactions were done for every VH, Vk or VL prim-
ers. Because of similar primer sequence, some PCR
reactions showed unspecific amplification, which did
not influence cloning (Fig. 3). PCR reactions of the first
step which generated clear bands were submitted for
the second step with proximal reverse primer. Products
were separated in polyacrylamide gel, marked bands
of the right size were cut from the gel, DNA eluted and
sequenced. Sequence finally proved which V-region
corresponds to expressed immunoglobulin. For Daudi
cell line, immunoglobulin heavy chain was VH3, and
kappa chain was Vk1. Eluted DNA of the right sequence
was used as a template for assembling of scFv.
VH
1-
Cμ
VH
2-
Cμ
VH
3-
Cμ
VH
4-
Cμ
VH
5-
Cμ
VH
6-
Cμ
Vk
1-
Cκ
Vk
2/
3-
Cκ
1500
500
300
100
1000
750
1500
500
300
100
1000
750
VH3-5’XbaI:
IgκC_stop_NotI
VH3-5’XbaI:
IgκC-SalI
Fig. 3. A. Agarose gel identification of the first step PCR products
of IgH and IgK variable regions semi-nested PCR amplification
of for Daudi cell line. B. Assembling of scFv by OE-PCR for Daudi
cell line in triplicates; primers for cloning are indicated.
Assembling and cloning of scFv. Two variable
fragments of heavy and light immunoglobulin chains
was further fused together by 6-histidine linker and
cloned by two different restriction sites into the ex-
pression vector. For that purpose, both fragments
were reamplified with primers including restriction
sites, start- and stop-codons and 6His-tag. Restriction
sites were chosen according to their presence in the
MCS of pET24b vector and absence in the consensus
regions of used immunoglobulin genes parts. The
last principle was not fully applicable due to non-
predictable sequence of junctional (CDR3) regions
and after hypermutation throughout V-regions. To the
beginning of construction the site for NdeI restrictase
containing start-codon (CATATG) was added. The end
of scFv was designed in two alternative ways. First,
the sequence of scFv in open frame finished with SalI
or HindIII restriction site. This site may be used to add
co-stimulator gene in fusion to scFv as SalI/HindIII —
NotI fragment. If no fusion co-stimulator gene is used,
transcription will terminate at the pET24b internal
stop codon after addition of another 6-His tag to the
expressing protein. Second, the sequence of scFv
ends with stop-codon and NotI site. Design of several
alternative restriction sites was advi sedly redundant for
a case of casual appearance on of the restriction site
sequence inside the segments and to make it possible
to clone construction in other vectors. Overall scheme
of genetic construction is shown on Fig. 4.
Overlap extension PCR (OE-PCR) was used
to fuse variable fragments of heavy and light immuno-
globulin chains. For that, 6 histidine codons — CAC-
CATCATCATCACCAC were added to 5’end of V-clon
primers of IgK/IgL and reverse complement sequence
to the 5’-end of C-clon primers of IgH. We designed
different His codons to avoid overlap shift during OE-
PCR. The sequences of cloning primers are shown
in Table 2.
Each of the purified DNA fragments from the previ-
ous step was amplified with appropriate pair of clon-
ing primers. Identical quantity of both PCR products
was mixed and subjected to 10 cycles of PCR without
primers, applying annealing temperature 55 °C and
high-fidelity polymerase. Immediately afterwards an-
other part of PCR mix containing flanking primers and
20 cycles of PCR proceeded with Tan= 65 °C. Fused
scFv construct was separated from non-used frag-
Fig. 4. Scheme of scFv constructions. Restriction site 1 (Rs1) — NdeI, Rs2 — SalI or HindIII, Rs3 — NotI
12 Experimental Oncology 35, 8–14, 2013 (March)
ments in agarose gel (Fig. 3 b), bands of DNA were
excised, eluted and directly cloned to pTZ57R/T vector.
After proving of the right sequence, scFv was sub-
cloned into pET24b by restriction and ligation.
Table 2. Sequences of primers for scFv assembling and cloning
Forward cloning primers
VH1-5’-XbaI GCG TCT AGA CAT ATG CAG GTG CAG CTG GTG CAG TCT GG
VH2-5’-XbaI GCG TCT AGA CAT ATG CAG GTC ACC TTG AAG GAG TCT GG
VH3-5’-XbaI GCG TCT AGA CAT ATG GAG GTG CAG CTG GTG GAG TCT GG
VH4-5’-XbaI GCG TCT AGA CAT ATG CAG GTG CAG CTG CAG GAG TCG GG
VH5-5’-XbaI GCG TCT AGA CAT ATG GAG GTG CAG CTG GTG CAG TCT GG
VH6-5’-XbaI GCG TCT AGA CAT ATG CAG GTA CAG CTG CAG CAG TCA GG
Reverse open frame primers
IgκC-SalI GCG GTC GAC AGA TGG CGG GAA GAT GAA G
IgκC-HindIII GCG AAG CTT AGA TGG CGG GAA GAT GAA G
IGLC2-7_SalI GCG GTC GAC CGA GGG GGC AGC CTT GGG
IGLC2-7_
HindIII
GCG AAG CTT CGA GGG GGC AGC CTT GGG
IGLC1_SalI CGC GTC GAC AGT GAC CGT GGG GTT GGC CTT GGG
IGLC1_HindIII GCG AAG CTT AGT GAC CGT GGG GTT GGC CTT GGG
Reverse termination primers
IGLC2-7_
stop_NotI
GCG GC GGC CGC TTA CGA GGG GGC AGC CTT GGG
IGLC1_stop_
NotI
GCG GC GGC CGC TTA AGT GAC CGT GGG GTT GGC CTT GGG
IgκC_stop_
NotI
GCG GC GGC CGC TTA AGA TGG CGG GAA GAT GAA G
Forward 6-his tag primers
Vk1-5’tag CAC CAT CAT CAT CAC CAC GAC ATC CAG ATG ACC CAG TCT CC
Vk2/3-5’tag CAC CAT CAT CAT CAC CAC GAT ATT GTG ATG ACC CAG ACT CCA
VL1_(51)_tag CAC CAT CAT CAT CAC CAC CAG TCT GTG TTG ACG CAG CCG
VL1_(36-
47)_tag
CAC CAT CAT CAT CAC CAC TCT GTG CTG ACT CAGC CA
VL1_(40)_tag CAC CAT CAT CAT CAC CAC CAG TCT GTC GTG ACG CAG CCG
VL2-tag CAC CAT CAT CAT CAC CAC TCC GTG TCC GGG TCT CCT GGA
VL3-tag CAC CAT CAT CAT CAC CAC ACT CAG CCA CCC TCG GTG TCA GTG
VL4-tag CAC CAT CAT CAT CAC CAC TCC TCT GCC TCT GCT TCC CTG GGA
VL5-tag CAC CAT CAT CAT CAC CAC CAG CCT GTG CTG ACT CAG CC
Reverse 6-his tag primers
Cg-tag GTG GTG ATG ATG ATG GTG CAG GGG GAA GAC CGA TGG
Cμ-tag GTG GTG ATG ATG ATG GTG GGA GAC GAG GGG GAA AAG
PVXCP cloning primers
PVXCP-F-SalI GCG GTC GAC ATG TCA GCA CCA GCT AGC ACA A
PVXCP-F-
HindIII
GCG AAG CTT ATG TCA GCA CCA GCT AGC ACA A
PVXCP-R-
NotI
G CGC GGC CGC TTA TGG TGG TGG TAG AGT GAC AAC
For cell lines Daudi, IM-9, RPMI1788 and patients
scFv was clones as NdeI — HindIII fragments with
additional plasmid 6-His tag at the C-end of protein.
Construction of scFv failed for RPMI1788 cell line
because of appearance of NdeI restriction site in-
side IGLV2–23 gene segment. For the last cell line,
Namalva, two kinds of construction were made. First
ended with the SalI restriction site without stop-codon,
which result in addition of 6-His at the 3’-end of scFv,
and second terminated close to the NotI site. We also
constructed chimeric construction of patient’s 1 id-
iotype and co-stimulating gene of Potato virus X coat
protein (PVXCP) [25] (Table 2).
Expression and purification of idiotype pro-
tein. Totally, six idiotype proteins were expressed
and purified as described in Materials and Methods.
Several experiments were employed to optimize
induction of protein expression with IPTG. Optimal
expression was detected after induction of E. coli
with 0.4 — 0.5 mM IPTG for 3 hours. SDS-PAGE
of cellular protein extracts demonstrated appearance
of clear band with an expected molecular weight
of 28–30 kDa (Fig. 5).
Our preliminary experiments showed that purifi-
cation of proteins on Ni-NTA column binding in non-
denaturing conditions was confronted with some prob-
lems: infirm binding of protein in the column and heavy
contamination of eluted fraction with high-molecular
weight bacterial proteins. Chromatography of idiotype
proteins in denaturing conditions (6M urea) provided
firm and specific binding to the Ni-NTA columns and
very reliable purification of target protein. All proteins
except form scFv went through the column, and after
washing, target protein was eluted.
— 50 kDa
— 30 kDa
— 20 kDa
28 kDa —
1 2 3
Fig. 5. Induction of Daudi-scFv protein expression in E. coli
induced by IPTG. 1 — before induction; 2 — after induction;
3 — molecular weight marker
We compared expression of two scFv constructs
of Namalva. First, encoding 263 amino acids, was
cloned as NdeI-stop-NotI fragment. Second, encoding
278 amino acids, was cloned as NdeI-SalI fragment
and included N-tail 6-His tag encoded by the vector.
Both proteins were successfully purified (Fig. 6), which
means that additional N-terminate 6-His tag did not
influence purification efficacy and can be removed.
Idiotype proteins were also identified by Western blot
analysis using antibodies to 6His-tag. Testing of id-
iotype protein for IM-9 cell line showed stained band
of the right size 28 kDa (Fig. 7).
— 50 kDa
— 30 kDa
— 40 kDa
— 25 kDa
— 28 kDa
— 15 kDa
— 10 kDa
1 4 72 5 83 6 99 10 11
Fig. 6. Fractions of Namalva scFv protein purification. 1 — cell
after induction; 2 — supernatant of cell lysate; 3 — lysate passed
through the column; 4 — wash of Ni-NTA columns with 20 mM im-
idazole; 5–8 — fractions of protein elution with 300 mM imidazole;
9 — molecular weight marker; 10 — pure scFv protein (SalI-
6-His); 11 — pure scFv protein (stop-NotI)
Experimental Oncology 35, 8–14, 2013 (March) 13
— 17 kDa
30 kDa —
25 kDa —
20 kDa —
15 kDa —
1 2 43 5
Fig. 7. Western blot of idiotype protein for IM-9 cell line. Lines:
1 — IM-9 protein + anti-6His-AB 1:1000; 2 — IM-9 protein + anti-
6His-AB 1:500; 3 — IM-9 protein + mouse serum; 4 — control
protein + anti-6His-AB 1:1000; 5 — control protein + mouse serum
DISCUSSION
Series of studies and clinical trials have provided
convincing evidence of biological and clinical efficacy
as well as of clinical benefit of soluble scFv protein
for anti-idiotype vaccination [3, 13, 26, 27]. How-
ever, three Phase III randomized clinical trials have
failed to achieve their main end points, presumably
because of imperfect study design or its conduction
[28–30]. At present, customized idiotypic vaccination
yet remains a non-approved, experimental approach
for patients with lymphoma. Taking into account that
hybridoma-derived Id-protein is time consuming and
difficult to produce new trials should probably employ
‘second-generation vaccine’ including recombinant
protein, fusion to gene-costimulator and usage
of DC for vaccine delivery [4, 28].
The aim of this study was development of a method
for the simplest, fast idiotype vaccine production, fea-
sible in basic laboratory of medical centre. Besides the
labor and time criteria, method of vaccine production
must be applicable to a wide range of patients and dif-
ferent mature B-cell malignances. To realize this aim,
two main difficulties have to be overcome: cloning
of individual, highly diverse immunoglobulin variable
fragments and convenient expression system with
protein purification.
Primers were designed for PCR amplification of four
immunoglobulin chains: immunoglobulin M and G heavy
chains and kappa- and lambda light chains to cover
most (>95%) B-cell lymphomas [23]. cDNA was used
as a template to allow application of reverse primers
to the proximal part of constant region, which possible
only after RNA splicing and make it possible to unify
primer set. We refused anchored PCR as a complication
of the method. Leader sequence of V-segment is not
required in bacterial expression system, and does not
contain essential antigen determinants for vaccine.
Start codon is included in primers for the cloning. Po-
sitions of the primers make it possible to amplify the
whole sequence of variable regions without loss or ad-
dition of amino acids except from 6His-tag in the middle
of construction. The use of consensus reverse primer
and similarity between forward (V-specific) primers
gives some nonspecific amplification, originating from
cross-annealing of V-specific primers. But, two steps
of PCR, heteroduplex analysis followed by purification
of DNA fragment in polyacrylamide gel and sequenc-
ing make identification of clonal rearrangements very
reliable, even if no data about immunoglobulin expres-
sion was obtained by immunophenotyping. Eluted from
gel DNA bands was used for amplification with cloning
and 6-His tagged primers, accomplishing preparation
of both fragments of scFv with 3 rounds of PCR.
Protein produced by hybridoma, transfected mam-
malian or insect cells takes month or more to prepare.
Therefore, choice of the expression system, providing
abundant, inexpensive and easily purified vaccine
was between bacteria and plants. Attractive method
of utilizing tobacco plants for individualized idiotype
vaccine production was proposed by A. McCormick
and colleagues [6–8, 31]. However, not all clinical
laboratories have facilities for plants culturing, and this
method require additional 2 weeks for plant infection
and accumulation of protein [6].
Expression of protein in bacteria may by the most
suitable method, but it also have some serious draw-
backs. First, there is some uncertainty if protein of pro-
karyotic expression is correctly folded and if absence
of glycosylation and an inability to form stable disulfide
bonds may influence antigen properties of idiotype.
Retention of original protein structure depends on the
methods of purification. In this study, we made certain
that denaturation of protein is necessary for chromato-
graphic purification. In denaturing conditions original
bacterial tertiary structure of protein was destroyed
and protein was refolded during dialysis. If there is any
influence of idiotypic proteins conformation their ability
to act as an antigen will be defined after immunological
assay of these vaccines. There are some evidences, that
scFv proteins expressed in bacteria retain the antigen-
binding capacity of original cell-born antibody [32, 21].
However, regardless of tertiary structure of expressed
and purified protein, it may be used for loading of DC,
as we are going to do in our further experience of vac-
cine testing and clinical trial. Second problem with
expression of protein in bacteria is that protein may
accumulate in insoluble form of inclusion bodies. It may
be solved by denaturing of protein and its refolding
during dialysis. Some studies described that accumula-
tion of allogenic protein in inclusion bodies in bacteria
is provoked by overexpression of protein and increased
temperature [33, 34]. In our experience that problem
was not essential and all proteins were soluble.
In conclusion, we elaborated rapid method of the
idiotypic vaccine production by PCR-based cloning
and expression of scFv in E. coli. Our study was initially
intended for DC loading with E. coli derived protein,
as the most safe and effective way of anti-idiotype im-
munization.
14 Experimental Oncology 35, 8–14, 2013 (March)
ACKNOWLEDGMENTS
This work was supported by the Health Ministry
of Belarus (Grant # 20101848) and The Foundation
for Fundamental Research, National Academy of Sci-
ences of Belarus (Grunt # 20081881).
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