Original Paper
|
|
|
file on Synergy |
Acta Biochim Biophys
Sin 2008, 40: 111-115
doi:10.1111/j.1745-7270.2008.00381.x
improved method to raise polyclonal
antibody using enhanced green fluorescent protein transgenic mice
Jianke
Ren1,2, Long Wang3, Guoxiang Liu1,2,
Wen Zhang1, Zhejin Sheng3,4, Zhugang Wang3,
Jian Fei3,4*
1 Laboratory of Molecular Cell Biology, Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of
Sciences, Shanghai 200031, China
2 Graduate School of Chinese Academy of Sciences,
Beijing 100049, China
3 Shanghai Nan Fang Model Organism Research Center,
Shanghai 201203, China
4 School of Life Science and Technology, Tongji
University, Shanghai 200092, China
Received: September
12, 2007
Accepted: October 29, 2007
This work was supported
by the grants form the E-Institutes of Shanghai Municipal Education Commission
(E03003) and the Science and Technology Commission of Shanghai Municipality
(06DZ19004, 06XD14014, 03DJ14088)
*Corresponding
author: 86-21-65980334; Fax, 86-21-65982429; E-mail, [email protected]
Recombinant
fusion protein is widely used as an antigen to raise antibodies against the
epitope of a target protein. However, the concomitant anticarrier antibody in
resulting antiserum reduces the production of the desired antibody and brings
about unwanted non-specific immune reactions. It is proposed that the carrier
protein transgenic animal could be used to solve this problem. To validate this
hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were
produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed
that the antiserum from the transgenic mice had higher titer antibody against
His6HA tag and lower titer antibody against EGFP compared with that from
wild-type mice. Therefore, this report describes an improved method to raise
high titer antipeptide polyclonal antibody using EGFP transgenic mice that
could have application potential in antibody preparation.
Keywords transgenic mice;
polyclonal antibody; EGFP
Antibodies are widely used in life science research and clinical
applications. In raising antibodies, antigen is often prepared by coupling the
antigen peptide to a carrier protein, such as keyhole limpet hemocyanin [15] or
bovine serum albumin [6,7]. In this case, the immunogenicity of the short
target peptide is enhanced due to the enlargement of the antigen molecule, but
it is a laborious process. Recombinant fusion proteins, using maltose-binding
protein [8,9] and glutathione S-transferase (GST) [1014] as the carrier
proteins, have also been adopted to raise antibody. The fusion proteins are
easy to acquire with recombinant DNA technology and purified by
well-established affinity chromatography methods. However, the majority of
antibody components in the antiserum generated by these methods are against
the carrier proteins [15,16]. Therefore, the titer of the desired antibody is
often low and a non-specific effect might exist. To avoid this problem, the
synthetic high-density multiple antigenic peptide system has been applied to
raise antipeptide antibody [17,18]. Although there is no anticarrier antibody
in the antiserum, the use of the multiple antigenic peptide system is limited
to very short peptides because of the space tendency of the branched multiple
antigenic peptides [19].It is well known that immune tolerance is established during
development in animals [20], so the antibodies generated against endogenous
proteins are abolished. The nature of this process implies that the production
of anticarrier antibody could be reduced after immunization with the
corresponding fusion protein, if the carrier protein was considered as an
endogenous product through a transgenic approach. To validate this hypothesis, we produced enhanced green fluorescent
protein (EGFP) transgenic mice as immune animals and chose His6HAtag as the
polypeptide epitope. After immunization with the fusion protein His6HAtag-EGFP,
the antisera from immunized mice were characterized by the titer of anti-EGFP
and anti-His6HA tag. Our data indicated that the immunized EGFP transgenic mice
produced higher titer antibody against His6HA tag and lower titer antibody
against the carrier protein EGFP compared with wild-type mice.
Materials and methods
Generation of EGFP transgenic
mice
To generate EGFP transgenic mice, plasmid pEGFP-N2 (BD Biosciences
Clontech, Heidelberg, Germany) was digested with PvuII, and the
resulting 4.1 kb fragment was purified and injected into fertilized mouse eggs
(C57Bl/6?CBA). The injected eggs were then
implanted into oviducts of pseudo-pregnant mice for continued development. The
offspring were screened for integration of the EGFP gene by polymerase chain
reaction (PCR) analysis of their tail DNA. Mice were kept in the conventional
animal facility of Shanghai Nan Fang Model Organism Research Center (Shanghai,
China) in accordance with institutional guidelines. Transgenic mice were bred
with C57Bl/6 mice and transmitted the EGFP gene for seven generations.
Construction of plasmids
Plasmid p6HHA-EGFP (kindly provided by Dr. GM Cheng of the Shanghai
Institutes for Biological Sciences, Shanghai, China) is a T7 promoter-based
vector that is used for expression of His6HAtag-EGFP in Escherichia coli.
In this vector, the His6HA tag is fused in frame to the N-terminus of the EGFP
coding sequence. The amino acid sequence of the His6HA tag is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSTMSGYPYDVPDYAGS
(51 amino acids). Plasmid p6HHA-EGFP was digested with NcoI and SacI
to remove the EGFP coding sequence and replaced in frame with the GST coding
sequence, amplified by PCR from plasmid pGEX-3x (Amersham pharmacia Biotech, Uppsala, Sweden)
using 5‘-primer 5‘-CCTACCATGGCCCCTATACTAGGTTATTG-3‘
and 3‘-primer 5‘-GTAGGAGCTCGATGAATTCCCGGGGAT-3‘. The
resulting plasmid was designated p6HHA-GST.Plasmid pGEXA was constructed previously in our laboratory by
adding multiple cloning sites between BamHI and EcoRI sites of
pGEX-3x. The DNA fragment encoding the EGFP protein was amplified by PCR from
plasmid pEGFP-N1 (BD Biosciences Clontech) using 5‘-primer 5‘-GGTAGCGGCCGCATGGTGAGCAAGGGCGAGG-3‘
and 3‘-primer 5‘-GTTTGAATTCTTTACTTGTACAGCTCGTCC-3‘.
The PCR product was digested with NotI and EcoRI, then inserted
into the NotI-EcoRI sites of pGEXA to construct plasmid
pGST-EGFP. The constructs was confirmed by DNA sequencing.
Expression and purification of
proteins
Escherichia coli BL21(DE3) was
transformed with the expression vectors p6HHA-EGFP, p6HHA-GST, and pGST-EGFP,
then the transformed cells were grown in Luria-Bertani medium containing 100 mg/ml ampicillin.
On reaching mid-log phase, the culture was induced with 0.8 mM isopropyl –D-thiogalactopyranoside
at 37 ?C for 4 h. The cells were harvested by centrifugation and disrupted by
sonication in phosphate-buffered saline (PBS) solution. The cytoplasmic extract
was filtered and subjected to affinity chromatography. His6HAtag-EGFP and
His6HAtag-GST were purified by a nickel affinity resin column (Anxin, Shanghai,
China) and GST-EGFP was purified by a glutathione Sepharose 4B column (Amersham
pharmacia Biotech) according to
the manufacturers instructions.Escherichia coli BL21(DE3) was
transformed with the expression vectors p6HHA-EGFP, p6HHA-GST, and pGST-EGFP,
then the transformed cells were grown in Luria-Bertani medium containing 100 mg/ml ampicillin.
On reaching mid-log phase, the culture was induced with 0.8 mM isopropyl –D-thiogalactopyranoside
at 37 ?C for 4 h. The cells were harvested by centrifugation and disrupted by
sonication in phosphate-buffered saline (PBS) solution. The cytoplasmic extract
was filtered and subjected to affinity chromatography. His6HAtag-EGFP and
His6HAtag-GST were purified by a nickel affinity resin column (Anxin, Shanghai,
China) and GST-EGFP was purified by a glutathione Sepharose 4B column (Amersham
pharmacia Biotech) according to
the manufacturers instructions.
Generation of polyclonal
antibody
Mice were divided into two groups, EGFP transgenic mice and
wild-type littermates. Each group contained five 8-week-old female mice. The
antigen (His6HAtag-EGFP) was dissolved in PBS solution at a concentration of
160 mg/ml and emulsified with an equal volume of complete Freund’s
adjuvant (Sigma-Aldrich, St. Louis, USA). At initial immunization, each mouse
was injected subcutaneously at several sites on the back with 40 mg immunogen in a
total volume of 500 ml. Two and 4 weeks later, the mice received two booster injections
intraperitoneally with the same amount of immunogen as the initial immunization
except that it was emulsified with incomplete Freund? adjuvant (Sigma-Aldrich).
After another 2 weeks, the mice were given an intravenous injection of 5 mg immunogen
without adjuvant. Three days later, the mice were bled from the retro-orbital
plexus to obtain antisera.
Enzyme-linked immunosorbent
assay (ELISA)
Ninety-six-well polystyrene microtiter plates were coated with 100 ml/well purified
protein solution at 1 mg/ml overnight at 4 ?C. Plates were blocked with 4% skim milk in
PBS for 2 h at 37 ?C. After the plates were washed three times with PBS, each
well was incubated with 100 ml diluted sera (dilution of 1:200, 1:400, 1:800, 1:1600, 1:3200,
…, 1:25,600) for 60 min at 37 ?C. Plates were washed four times with PBS-T
(PBS and 0.1% Tween-20), and each well was incubated with 100 ml anti-mouse
immunoglobulin G-horseradish peroxidase conjugate (1:7000 dilution;
Sigma-Aldrich) for 60 min at 37 ?C. Plates were washed five times with PBS-T and
100 ml tetramethylbenzidin substrate solution was added to each well.
After incubation at room temperature for 10 min, the reaction was stopped with
the addition of 25 ml of 2 M H2SO4 into each well. The
absorbances at 450 nm were measured with an ELISA plate reader (Bio-Rad,
Hercules, USA). The titer of antibody was defined as the serum dilution at
which the absorbance at 450 nm was half of the maximal value.
Western blot analysis
Ten nanograms of purified GST-EGFP or His6HAtag-GST were electrophoresed
in 15% sodium dodecyl sulfate-polyacrylamide gel. The samples were transferred
to nitrocellulose membranes for 60 min at 100 V. The membranes were then
rinsed with Tris-buffered saline Tween 20 (TBST) and blocked with 5% skim milk
for 60 min. One membrane was incubated with antiserum (1:7000 dilution) from
immunized transgenic mice and the other was incubated with antiserum (1:7000
dilution) from immunized wild-type mice in parallel for 60 min. Both membranes
were then washed four times with TBST and incubated with goat anti-mouse
immunoglobulin G-horseradish peroxidase conjugate (1:7000 dilution;
Sigma-Aldrich) for 60 min and washed in TBST. Membranes were finally developed
with enhanced chemiluminescence detection reagents (Sigma-Aldrich) and detected
under the same conditions.
Results
Generation of EGFP transgenic
mice
After screening of the offspring mice by PCR identification [Fig.
1(A)], the stable expression of EGFP in transgenic mice was confirmed by the
detection of green fluorescence in live mice with ultraviolet excitation
light. The high level of EGFP expression that was directed by a strong
cytomegalovirus promoter could be detected in the transgenic newborns after
the transgenic mice were bred with C57Bl/6 mice for seven generations [Fig.
1(B)]. Immunohistochemistry analysis with anti-EGFP antibody also showed
the ubiquitous expression of EGFP in transgenic mice (data not shown).
Construction of plasmids,
expression and purification of fusion proteins
In order to produce the antigen, the recombinant plasmid p6HHA-EGFP
was transformed into E. coli BL21(DE3) to express fusion protein
His6HAtag-EGFP, used to immunize both transgenic and wild-type mice. The
purified proteins His6HAtag-GST and GST-EGFP were used for detection of the
antibody titer against His6HAtag and EGFP [Fig. 2(A)]. The corresponding
plasmids were named p6HHA-GST and pGST-EGFP, respectively.The purified fusion proteins were separated by 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis [Fig. 2(B)]. The purity of
the proteins was estimated to be approximately 90% for His6HAtag-EGFP and 85%
for His6HAtag-GST.
EGFP transgenic mice generated
higher titer polyclonal antibody against His6HAtag with a reduced titer of anti-EGFP
EGFP is the auto-antigen for EGFP transgenic mice, as pre-immune
sera from EGFP transgenic mice did not give positive reactions to GST-EGFP
(data not shown). After immunization with purified protein His6HAtag-EGFP, the
antisera from EGFP transgenic mice and wild-type mice were titered against
His6HAtag-GST and GST-EGFP, respectively, by ELISA. The results showed
that the titer of the antiserum against His6HAtag-GST protein was raised to
approximately 5300 for EGFP transgenic mice, and to approximately 2600 for
wild-type mice [Fig. 3(A)]. As for the titer of antiserum against
GST-EGFP protein, EGFP transgenic mice showed the average titer of 6500,
whereas wild-type mice showed a titer of 15,000 [Fig. 3(B)]. The
target/carrier ratio (the antiserum titer of anti-His6HAtag to that of
anti-EGFP protein) of the transgenic mice was significantly higher than that of
wild-type mice [Fig. 3(C)].Furthermore, western
blot analysis showed that the antiserum from EGFP transgenic mice displayed a
better detection sensitivity to His6HAtag-GST [Fig. 4(A)] and less
sensitivity to GST-EGFP [Fig. 4(B)] than that from wild-type mice. These
results suggested that by immunization of fusion protein His6HAtag-EGFP, the
EGFP transgenic mice generated higher titer polyclonal antibody against His6HA
tag with lower titer of anti-EGFP antibody.
Discussion
In this study, we showed that EGFP transgenic mice could be used to
raise high titer polyclonal antibodies against a foreign tag using EGFP fusion protein
as the antigen. The antiserum from transgenic mice contained higher titer of
antibody against the target peptide and lower titer of antibody against the
carrier protein compared with that from wild-type mice. However, the titer of
the antibody against the epitope, raised in the transgenic mice, was only twice
that obtained in the wild-type mice, whereas the target/carrier ratio (the
antiserum titer of antitarget peptide to that of anticarrier protein) of the
antibody from transgenic mice was approximately 4-fold of that from wild-type
mice [Fig. 3(C)]. EGFP could also be used as a protein fusion partner to
monitor and optimize the production and purification of recombinant protein by
the green fluorescence emission.Significant titer against the carrier protein (EGFP) was
nevertheless obtained in EGFP transgenic mice. This might be the result of
using intensified immunization strategies that broke the immune tolerance.
Another possible explanation is that EGFP transgenic mice produced antibodies against
denatured EGFP protein during immunization. In addition, complete Freund’s
adjuvant might have partially denatured the immunogen.The
increase of the absolute titer of antibody against the tags in this study was
limited and the improvement of the specificity of the antibody against the
target tag requires substantiation with further evidence, however, the
significant increase of the target:carrier ratio of the antibody titer in
transgenic mice suggested that this method is valuable. This strategy might
also be suitable for other fusion protein systems, such as GST and
maltose-binding protein, if the corresponding transgenic mice are generated.
Furthermore, this method is not limited to the field of polyclonal antibody
preparation and might be more useful in monoclonal antibody development.
References
1 Lerner RA, Green N, Alexander H, Liu FT,
Sutcliffe JG, Shinnick TM. Chemically synthesized peptides predicted from the
nucleotide sequence of the hepatitis B virus genome elicit antibodies reactive
with the native envelope protein of Dane particles. Proc Natl Acad Sci USA
1981, 78: 3403–3407
2 Kirkley JE, Goldstein AL, Naylor PH. Effect
of peptide-carrier coupling on peptide-specific immune responses. Immunobiology
2001, 203: 601–615
3 Edwards RJ, Singleton AM, Murray BP, Davies
DS, Boobis AR. Short synthetic peptides exploited for reliable and specific
targeting of antibodies to the C-termini of cytochrome P450 enzymes. Biochem
Pharmacol 1995, 49: 39–47
4 Beekman NJ, Schaaper WM, Turkstra JA, Meloen
RH. Highly immunogenic and fully synthetic peptide-carrier constructs
targetting GnRH. Vaccine 1999, 17: 2043–2050
5 Riveros-Moreno V, Beddell C, Moncada S.
Nitric oxide synthase. Structural studies using anti-peptide antibodies. Eur J
Biochem 1993, 215: 801–808
6 Bernard D, Nicolas C, Maurizis JC, Betail G.
A new method of preparing hapten-carrier immunogens by coupling with Saccharomyces
cerevisiae by periodate oxidation. J Immunol Methods 1983, 61: 351–357
7 Hansen PR, Flyge H, Holm A, Lauritzen E,
Larsen BD. Photochemical conjugation of peptides to carrier proteins using
1,2,3-thiadiazole-4-carboxylic acid. Immunoreactivity of free C-terminal epitope
with specific antibodies. Int J Pept Protein Res 1996, 47: 419–426
8 Castrop J, Verbeek S, Hofhuis F, Clevers H.
Circumvention of tolerance for the nuclear T cell protein TCF-1 by immunization
of TCF-1 knock-out mice. Immunobiology 1995, 193: 281–287
9 Simabuco FM, Carromeu C, Farinha-Arcieri LE,
Tamura RE, Ventura AM. Production of polyclonal antibodies against the human
respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia
coli. Protein Expr Purif 2007, 53: 209–215
10 Ray MK, Wang G, Barrish J, Finegold MJ, DeMayo
FJ. Immunohistochemical localization of mouse Clara cell 10-KD protein using
antibodies raised against the recombinant protein. J Histochem Cytochem 1996,
44: 919–927
11 Marikar FM, Sun QM, Hua ZC. Production of the
polyclonal anti-human metallothionein 2A antibody with recombinant protein
technology. Acta Biochim Biophys Sin 2006, 38: 305–309
12 Yu H, Yang Y, Zhang W, Xie YH, Qin J, Wang Y,
Zheng HB et al. Expression and purification of recombinant SARS coronavirus
spike protein. Acta Biochim Biophys Sin 2003, 35: 774–778
13 Song BL, Qi W, Wang CH, Yang JB, Yang XY, Lin
ZX, Li BL. Preparation of an anti-Cdx-2 antibody for analysis of different
species Cdx-2 binding to acat2 promoter. Acta Biochim Biophys Sin 2003, 35: 6–12
14 Hawkins NC, Ellis GC, Bowerman B, Garriga G.
MOM-5 frizzled regulates the distribution of DSH-2 to control C. elegans
asymmetric neuroblast divisions. Dev Biol 2005, 284: 246–259
15 Gearing AJ, Bird CR, Callus M, Thorpe R. The
effect of primary immunization and concanavalin A on the production of
monoclonal natural antibodies. Hybridoma 1986, 5: 243–247
16 Peeters JM, Hazendonk TG, Beuvery EC, Tesser
GI. Comparison of four bifunctional reagents for coupling peptides to proteins
and the effect of the three moieties on the immunogenicity of the conjugates. J
Immunol Methods 1989, 120: 133–143
17 Tam JP. Synthetic peptide vaccine design:
Synthesis and properties of a high-density multiple antigenic peptide system.
Proc Natl Acad Sci USA 1988, 85: 5409–5413
18 Gomara MJ, Riedemann S, Vega I, Ibarra H,
Ercilla G, Haro I. Use of linear and multiple antigenic peptides in the
immunodiagnosis of acute hepatitis A virus infection. J Immunol Methods 2000,
234: 23–34
19 Tam JP, Clavijo P, Lu YA, Nussenzweig V,
Nussenzweig R, Zavala F. Incorporation of T and B epitopes of the
circumsporozoite protein in a chemically defined synthetic vaccine against
malaria. J Exp Med 1990, 171: 299–306
20 Liacopoulos P, Halpern BN, Neveu T. Immune
tolerance: Its mechanisms and its specificity. Rev Fr Etud Clin Biol 1964, 22:
118–125