全 文 ：ISSN　1007-7626
CN　11-3870 Q
中国生物化学与分子生物学报
Chinese Journal of Biochemistry and Molecular Biology
2007年 9 月
23(9):730 ～ 737
Effect of Displaying P277 Peptide on Surface of
L-asparaginase on Its Antigenicity and Efficacy
of Autoimmune Diabetes Prevention in NOD Mice
ZHU Ai-Hua1), 2) , LIU Wen-Tao1) , Long Jun1) , Wu Jie1) , LIU Jing-Jing1) , LI Tai-Ming1)＊
(1)Minigene Pharmacy Laboratory , School of Life Science &Technology , China Pharmaceutical University , Nanjing 210009 , China;
2)School of Life Science , Xuzhou Normal University , Xuzhou 221116 , Jiangsu , China)
Abstract　The recombinant chimeric enzyme of AnsB-TTP-P277 comprising L-asparaginase , a tetanus toxin
peptide spacer and P277 was expressed as a soluble protein in Escherichia coli.The purified chimeric enzyme
exhibited primary activity of the native asparaginase.Prediabetic NOD mice immunized with the chimeric
enzyme could induce specific antibodies against P277 and the specificity of anti-P277 antibodies was verified by
Western blot assay.The study showed that displaying the P277 epitope on the surface of asparaginase could
effectively overcome the weak antigenicity of the P277 epitope and evoke a strong P277-specific immune
response in mice.Moreover , the concentration of blood glucose was measured by an automated analyzer.
Histochemical analysis of mice pancreas tissues showed that the administration of the chimeric enzyme to NOD
mice could prevent the development of diabetes more efficiently than the peptide P277 itself.
Key words　L-asparaginase;P277 peptide;insulin-dependent diabetes mellitus;antibody;blood glucose
L-天冬酰胺酶表面呈现对 P277肽免疫原性及
抗 NOD小鼠糖尿病作用的影响
朱爱华1), 2) , 　刘文涛1) , 　龙　军1) , 　吴　洁1) , 　刘景晶1) , 　李泰明1)＊
(1)中国药科大学生命科学与技术学院微基因药物实验室 , 南京　210009;2)徐州师范大学生命科学学院 , 江苏 徐州　221116)
摘要　将 P277多肽融合在 L-天冬酰胺酶的 C 端 ,中间引入破伤毒素肽(TTP),重组嵌合酶 AnsB-
TTP-P277能以可溶性蛋白的形式表达于大肠杆菌周质内 ,且能保持大约 80%的天然酶催化活力.
用纯化的嵌合酶腹腔注射非肥胖性(NOD)小鼠 , 可成功诱导小鼠体内产生高水平抗 P277抗体.研
究表明 ,P277肽可呈现于 L-天冬酰胺酶表面 ,有效地克服单独 P277的弱免疫原性 ,激发机体产生
较强烈的免疫反应.小鼠胰腺病理学切片也显示:重组嵌合酶 AnsB-TTP-P277和单纯 P277肽相比 ,
能更有效地抑制 NOD小鼠糖尿病的发生.
关键词　L-天冬酰胺酶;P277肽;胰岛素依赖型糖尿病;抗体;血糖
中图分类号　Q789;R392
Received:Apri l 2 , 2007;Accepted:June 20 , 2007
Supported by National High Technology Research andDevelopment Program of
China(863 Program , No.2002 AA217031-2)and National Natural Science
Fundation of China(No.30500458, 30672464)
＊Corresponding author　Tel:025-83271346;
E-mail:taimingli@163.com
收稿日期:2007-04-02 ,接受日期:2007-06-20
国家高技术研究发展计划(863计划 , No.2002 AA217031-2)和国家自
然科学基金(No.30500458 , 30672464)资助
＊联系人　Tel:025-83271346;E-mail:taimingli@163.com
　　It is now generally agreed that type 1 diabetes
mellitus , also called insulin-dependent diabetes mellitus ,
is often caused by an autoimmune process in which T cells
invade the pancreatic islet and destroy the insulin-
producing β cells[ 1] .The P277 peptide is composed of a
24-amino acid sequence from the 60 kD human heat shock
protein(hsp60)[ 2] .This peptide bears at least one T cell
epitope that is important for the development of diabetes.
Prediabetic non-obese diabetic(NOD) mice manifest
spontaneous antibody formation and T cell responses to
hsp60 and its peptide P277 fragment prior to the onset of
DOI :10.13865/j.cnki.cjbmb.2007.09.015
diabetes
[ 3 , 4] .A single administration of peptide P277 in
NOD mice could arrest the diabetogenic autoimmune
process , even in its advanced developing phase.[ 4 , 6 , 7] .
The favorable effect of P277 was associated with the
induction of specific antibodies of the IgG1 and IgG2b
isotypes , and a shift from Th1 type (IFN-γ)to Th2 type
cytokine (IL-4 , IL-10) secretion in the cytokine
production profile of the anti-P277 T cell population in
both obese and non-obese mice model systems[ 8 , 9] .
The human immune system can generate effective
immune responses against pathogens.However , little
response to common antigens , such as P277 , was
observed , as the lack of their presentation to the immune
system in a “dangerous” manner[ d2] .In order to overcome
the weak antigenicity of P277 alone , a recombinant
chimeric enzyme of AnsB-TTP-P277 was constructed , in
which asparaginase(AnsB), tetanus toxin peptide (TTP)
and P277 were fused together.TTP could bind most MHC
haplotypes and elicit strong T-cell help for B cells[ 10] .
Studies have shown that asparaginase monomers could
form tetramers , octamers , twelve-mers , sixteen-mers , or
other multiple-unit complexes[ 11] .When the chimeric
enzymes formulated into multiplexed molecules four ,
twelve , sixteen or more P277 epitopes could be presented
to antigen present cells (APCs).Meanwhile , the homo-
multimers of the chimeric enzyme , which possessed huge
molecular weight , could be easily and effectively captured
by APCs.Thus , strong immune responses against P277
would be evoked after vaccinating animals with the
chimeric enzyme in vivo.Therefore , the chimeric enzyme
of AnsB-TTP-P277 may be used as a potential vaccine for
preventing the progression of diabetes.
In this paper , a vector expressing chimeric enzyme
AnsB-TTP-P277 was constructed and transformed into E.
coli BL21 (DE3).Recombinant AnsB-TTP-P277 was
purified and then used to immunize NOD mice.Serum
samples from the immunized mice were collected and the
production of anti-P277 antibodies was tested.The blood
glucose concentration of mice was measured and diabetic
pathological analysis was performed.Our data indicated
that the rAnsB-TTP-P277 could promote the efficacy of
antibody induction than that of peptide P277 alone , hence
exhibited better autoimmune diabetes prevention effect in
NOD mice.
1　Materials and Methods
1.1　Materials
Recombinant human vascular endothelial growth
factor(rhVEGF)was a gift from Dr.L.Jingjing[ 12] .The
pED plasmid was provided by Dr.Z.Yankai[ 13] .The
pET28-AnsB-TTP plasmid and the purified native L-
asparaginase were gifts from Dr.Qi G F.[ 14] .All other
chemicals and reagents were obtained from the following
companies TaKaRa , Amresco , and Sigma.Four-week-old
female NOD Lt mice were provided by Shanghai Slaccas
Experiment Animal Limited Company .
1.2　Expression and purification of the peptide P277
The strategy used in the expression and purification
of the peptide P277 was identical to our previous
publications
[ 15-17] .The sequence encoding P277 was
synthesized by PCR and fused to the C-terminal sequence
of L-asparaginase , encompassing residues 199—326 ,
through an acid-labile aspartyl-prolyl linker. The
recombinant protein was expressed at high levels in E.
coli BL21 (DE3).The fusion proteins were purified to
homogeneity by means of cell disruption , washing the
inclusion bodies and ethanol precipitation.The P277 was
released from the fusion proteins by cleavage with
hydrochloric acid and purified by cellulose DE-52(Whatman , USA)column chromatography.
1.3 　 Construction of vectors and expression of
rAnsB-TTP-P277
The AnsB-TTP gene was amplified by PCR from the
plasmid pET28-AnsB-TTP using A1 (5′-TTC GCCATG
GCCAAGAACAATTGCGTA CG-3′) and A2 (5′-AAA
ATGATCAGTAGTACCAGAGATTAC-3′)as forward and
reverse primers , respectively.PCR was carried out in a
total 50 μl reaction mixture containing 100 pmol H1 , 10
pmol H2 , 100 ng plasmid DNA , 2 μl dNTP (10 μmol
ml)and 4 units of PFU DNA polymerase (Promega ,
USA).PCR conditions were as follows:denaturing at
94 ℃ for 60 s , annealing at 58 ℃ for 60 s , and
extension at 72 ℃ for 2.5 min for total 30 cycles.
Amplified AnsB-TTP DNA fragments were analyzed in
1.0%(W V) agarose gel.After purification , the
amplified fragments were digested by endonuclease Nco Ⅰ
and Bcl Ⅱ and then inserted into the pED-P277
previously digested by NcoⅠ and BamH Ⅰ to form a new
expression vector pET28-AnsB-TTP-P277.It contained
asparaginase , a 25 amino acid-residue T cell epitope
derived from tetanus toxin peptide (TTP)spanning amino
acids 831—854(QYIKANSKFIGITELIYSYFPSVI)and a
25-amino-acid sequence of P277 peptide(PVLGGGVALLRVIPALDSLTPANED).A linker peptide
of Ser-Gly-Thr was introduced between TTP and P277.
The pET28-AnsB-TTP-P277 vector was used for
transforming E.coli BL21 (DE3) and the resulting
colonies were screened by kanamycin resistance and PCR ,
then sequenced to confirm the correct insertion.
The E .coli BL21 (DE3) containing vector of
pET28-AnsB-TTP-P277 were cultured in 1 000 ml of
Luria-Bertani (LB)medium containing kanamycin (50
μg μl)at 37 ℃ and shaken at 100 r min.When the
optical density value at 600 nm (A600)of the culture
reached 0.5 , 10 ml lactose solution (500 mmol L)was
added into the medium to induce the expression of
chimeric enzyme.After 4 hours induction period , the
cultures were harvested by centrifugation at 4 ℃ for 5min
at 9 000 × g.The cells (wet weight of 10.0 g)were
resuspended in 100 ml of 10 mmol L phosphate buffer
731No.9 ZHU Ai-Hua et al:L-asparaginase display of P277 for autoimmune diabetes prevention 　
(pH 8.0)containing 1 mmol L EDTA , 40 μl lysozyme
(10μg ml)and 10μl DNase Ⅰ (1 mg ml)and shaken at
37 ℃for 30 min to lyse the cells , and the supernatant
and pellet were analyzed by 12%SDS-polyacrylamide gel
electrophoresis(PAGE).
1.4　Purification of the chimeric enzyme
Cells harboring pET28-AnsB-TTP-P277 were grown ,
induced and harvested as described above.The cell
lysates were centrifuged at 12 000 ×g for 10 min at
4 ℃, the supernatant was collected and then ammonium
sulfate was added into the supernatant until the saturation
of ammonium sulfate in the supernatant reached 30%.
The solution was kept at 4 ℃ for 1 hour and then the
sediment was collected by centrifugation at 8 000×g for
10 min at 4 ℃.Approximately 85% of the rAnsB-TTP-
P277 protein was precipitated.The precipitate was
solubilized in 100 ml of 10 mmol L phosphate buffer(pH
7.4)containing 1mmol L EDTA and dialyzed with 10
mmol L phosphate buffer (pH 7.4)for 24 hours.The
solution was loaded on a DE-52 cellulose column(Whatman , USA)which had been equilibrated with 10
mmol L phosphate buffer (pH 7.4).The column was
washed with 10 mmol L phosphate buffer (pH 7.4)until
the absorbance of elution at 280 nm was below 0.2.Then
the column was eluted with a linear gradient of 50 to 300
mmol L NaCl in 10 mmol L phosphate buffer(pH 7.4).
The fractions containing proteins were collected and
analyzed with a 12%SDS-PAGE.
1.5　Activity assay of chimeric enzyme
Activity of chimeric enzyme was detected by Nessler
reaction as described by Wriston with a slight
modification
[ 18] . Asparaginase could hydrolyze L-
asparagine into L-aspartate and ammonia , and the latter
could react with Nessler reagent to produce an orange or
red product.One unit(U)of enzyme activity was defined
as the amount of enzyme that liberated 1mmol of ammonia
from L-asparagine per min at 37 ℃.Activity of chimeric
enzyme was the units of enzyme activity of per milligram
of protein.25μl of purified rAnsB-TTP-P277(4 μg μl)
protein reacted with 25 μl of L-asparagine(0.4 mmol L)
in 50μl boric acid buffer(pH 8.0)at 37 ℃ for 15 min.
50 μl of 15%(W V)trichloroacetic acid was added into
the reaction solution to stop the reaction and Nessler
reagent was added.The optical density of the reaction
solution at 500 nm (A500)was measured.A standard
Nessler curve of ammonium sulfate was done by the above
method.The ammonium produced by the reaction was
counted according to the Nessler curve of ammonium
sulfate.At the same time purified native asparaginase
reacted with L-asparagine in the above conditions as a
control.
1.6　Vaccination
Four-week-old female NOD mice were divided into
four groups of ten animals each (n =10 per group).
Three groups , respectively , received three i.p.
inoculations of 50μg of purified AnsB-TTP-P277 , peptide
P277 or L-asparaginase in incomplete Freund′s adjuvant(Sigma)at 4 weeks , 7 weeks , and 10 weeks of age;the
control mice received three emulsion of phosphate-
buffered saline in incomplete Freund′s adjuvant.All mice
were bled to detect antibodies to P277 in the blood and
blood glucose at every month.
1.7　Blood Glucose
After the final administration serum samples were
collected at monthly interval.The concentration of blood
glucose was measured by Hitachi automatic analyzer
(model-7150 , Tokyo , Japan).A mouse was considered
to be diabetic if the blood glucose was >3 standard
deviations above the mean of that measured in no diabetic
or prediabetic control mice without insulitis , ≈11.1
mmol L or greater.
1.8　Expression and purification of rhVEGF-P277
The pET28-VEGF-P277 plasmid was constructed to
express the recombinant protein of rhVEGF-P277
containing VEGF (human vascular endothelial growth
factor 121)and P277.RhVEGF-P277 was used to detect
the presence of anti-P277 antibodies in enzyme-linked
immunosorbent assay (ELISA) and the specificity of
antibodies in Western blot assay.The VEGF gene was
amplified by PCR from the plasmid pCR3.1-Uni-VEGF
according to published sequence of VEGF using forward
primer(5′-TTT TCCATGGCAGAAGGAGGAGGG CAG-
3′)and reverse primer (5′-GGG GGGATCCAGTTTTT
CTTGTCTTGCTCTAT-3′)[ 12] .After purified and digested
with Nco Ⅰ and BamH Ⅰ the amplified VEGF fragment(365 bp)was inserted into the pED-P277 vector , which
was also digested by Nco Ⅰ and BamH Ⅰ , to form the
plasmid pET28-VEGF-P277.The plasmid pET28-VEGF-
P277 was transformed into E .coli BL21(DE3).
Cells containing the pET28-VEGF-P277 plasmid
were cultured , induced and collected as described above.
Cells(10 g of wet weight)were suspended in 100 ml of
10 mmol L phosphate buffer supplemented with 400 μl
lysozyme(10μg ml)and 100μl DNaseI (1 mg ml)and
then shaken at 200 r min for 1 hour at 37 ℃.The cells
were collected by centrifuge at 12 000×g for 15 min and
the cell pellets were washed twice with 2 mol L urea and
then solubilized in 4 mol L urea supplemented with 10
mmol L DL-dithiothreitol (DTT).The solution was
dialyzed with double-distilled H2O for 48 hours and then
loaded on a DE-52 column , which had been equilibrated
with 10 mmol L phosphate buffer (pH 7.4).The column
was washed with the 10 mmol L phosphate buffer (pH
7.4)until the absorbance value of elution at 280 nm was
below 0.2.The protein was eluted using a linear gradient
of 50 to 500 mmol L NaCl in 10 mmol L phosphate buffer
(pH 7.4).The fraction containing rhVEGF-P277 was
collected and analyzed by 15%SDS-PAGE gel.
1.9　ELISA analyses of specific antibodies against
P277
732 Chinese Journal of Biochemistry and Molecular Biology Vol.23
Purified rhVEGF-P277 was diluted with 0.1mmol L
carbonate-bicarbonate buffer (pH 9.5) and applied to
flat-bottom ELISA plates (Costar , USA)at 100 μg per
well.ELISA plates were incubated overnight at 4 ℃.
Wells were blocked with 10mmol L phosphate buffer(pH
7.4) containing 5% (W V) bovine serum albumin(Sigma , USA)for 1 hours.The wells were incubated
with 1∶100 two-fold dilutions of serum samples collected
from immunized animals overnight at 4 ℃.The wells
were washed three times with 10 mM phosphate buffer(pH 7.4)containing 0.1%(V V)Tween-20.Each well
was added 100 μl goat anti-mouse IgG horseradish
peroxidase which was diluted 1∶20 000 with 10 mmol L
phosphate buffer (pH 7.4)containing 1%(W V)BSA
and then incubated at 37 ℃ for 1 hour.The wells were
washed intensively six times with 10 mmol L phosphate
buffer(pH 7.4)containing 0.1%(V V)Tween-20.To
each well , 50 μl of 0.01% (W V) 3 , 3′, 5 , 5′-
tetramethylbenzidine and 50 μl 0.2 mol L Na2HPO4-0.1
mol L citrate buffer(pH 5.5)containing 0.24%(W V)
H2O2-urea was added and incubated at 37 ℃ for 15 min.
The reaction was stopped by adding 50 μl 2 mol L
H2SO4.A450 value of the reaction solution was measured
by Multiskan spectrum microplate spectrophotometer(Thermo , USA).
1.10 　Western blot analyses of specific antibodies
against P277
Purified rhVEGF-P277 and rhVEGF in the presence
or absence of DTT were ran in a 15% SDS-PAGE gel
under denaturing conditions and then transferred onto a
nitrocellulose membrane (Millipore , USA). The
membrane was blocked with Tris-buffered saline (TBS ,
pH 7.5;100 mmol L Tris-HCl , 0.9%(W V)NaCl)
containing 5%(W V)BSA for 2 hours at 37 ℃.Serum
samples from immunized mice were diluted 1∶50 in TBS
containing 5% (W V) BSA and then used for
hybridization with proteins on the membrane.The
membrane was washed four times with TTBS (TBS
containing 0.1% (V V)Tween-20).HRP-conjugated
rabbit anti-mouse IgG was diluted 1∶200 with TBS
containing 5%(W V)BSA and then reacted with the
membrane.After washing the membrane for four times
with TTBS the reaction was developed using 10 mmol L
Tris-HCl buffer (pH 7.5)containing 0.05%(W V)3 ,
3′-diaminobenzidine and 0.012%H2O2(V V)at 37 ℃
for 15 min.
1.11　Pancreas tissue histopathology
After being killed , pancreas of mice were removed
and fixed with 10% formalin solution.Formalin-fixed
paraffin blocks of pancreas tissue were sectioned with a
microtome , stained with hematoxylin and eosin , and
evaluated in a blinded fashion by a pathologist to
determine whether the vaccine cause any pathological
change.
2　Results
2.1　rAnsB-TTP-P277 was expressed as a soluble
periplasmic protein and exhibited high enzyme
activity
After precipitation with 30% saturated ammonium
sulfate the chimeric enzyme of AnsB-TTP-P277 was
separated.The precipitate was resolubilized in 10 mmol L
phosphate buffer (pH 7.4)and then dialyzed with 10
mmol L phosphate buffer(pH 7.4)to remove ammonium
sulfate.The chimeric enzyme was further purified by
DE52-cellulose and eluted at 80 to 120 mmol L NaCl in
10 mmol L phosphate buffer(pH 7.4).rAnsB-TTP-P277
was purified to approximate homogeneity using the above
two steps(Fig.2 A , lane 5).
The activity of native asparaginase and rAnsB-TTP-
P277 was measured by Nessler′s reaction.The result
showed that the activity of native asparaginase was 240 U
per mg of protein.The activity of rAnsB-TTP-P277 was
200 U per mg of protein.The chimeric enzyme
asparaginase still retained most(83%)of the activity.
Fig.1　Construction of recombinant chimeric enzymes
AnsB-C-P277 fusion protein(A), and AnsB-TTP-P277 fusion protein(B)were expressed in
E.coli using the pED or pET28 vector under the direction of the T7 promoter.The T7
promoters are represented in hatched boxes.The AnsB-C or AnsB gene is represented by open
box and the tetanus toxin 831-854 fragment (TTP)is drawn in black.In addition , the P277
and the TTP are represented by single-letter codes
733No.9 ZHU Ai-Hua et al:L-asparaginase display of P277 for autoimmune diabetes prevention 　
Fig.2　Expression and purification of rAnsB-TTP-P277
(A).RAnsB-TTP-P277 fusion protein was analyzed in 12% SDS-PAGE gel stained with Coomassie blue.Lane 1, Protein
molecular weight markers;Lane 2, Total proteins of E.coli BL21 (DE3) containing pET28-AnsB-TTP-P277 uninduced by
lactose;Lane 3 , Total proteins of E.coli BL21(DE3)containing pET28-ansB-TTP-P277 induced by lactose;Lane 4, The
sample of partially purified rAnsB-TTP-P277;Lane 5 , Purified rAnsB-TTP-P277;Lane 6, Purified L-asparaginase.
(B).Three-dimensional delineation of AnsB-TTP-P277 based on the publi shed crystal structure of E.coli asparaginase.
The white arrows showed 4 linear p277 peptide fragments displayed on the surface of tetra-unit asparaginase
2.2 　Specific anti-P277 antibody formed in mice
inoculated with the chimeric enzyme of AnsB-TTP-
P277
ELISA assay showed that the serum samples
collected from mice immunized with the chimeric enzyme
of AnsB-TTP-P277 contained anti-P277 antibodies.The
high level of anti-P277 antibodies could be detected as
early as the age of 7-week-old post the first immunization(Fig.3).Specific antibodies against P277 continued to
be made and last for more than fourteen weeks in vivo
even though animals were vaccinated with the chimeric
enzyme for only three times.The titer of anti-P277
antibodies of NOD mice vaccined with AnsB-TTP-P277
was higher than with the peptide P277 only (Fig.3).
VEGF could form a homodimeric protein by
intermolecular disulfide bonds
[ 13] . Therefore when
analyzed by 15% SDS-PAGE gel VEGF would form a
single band if the sample buffer contained reductant such
as DL-dithiothreitol (DTT).If the sample buffer didn′t
contain reductant VEGF would form two bands.In
Western blot assay this characteristic of rhVEGF-P277
would help to detect specific antibodies against P277.The
result showed that in-frame fusion of P277 with VEGF did
not prevent VEGF from forming homodimeric fusion
protein.It was verified that the band of the monomeric
fusion protein of rhVEGF-P277 “shifted” after folding into
a higher molecular weight dimer under non-reducing
condition.
Fig.4 showed that sera from rAnsB-TTP-P277
immunized mice bound the rhVEGF-P277 monomer(lane
3)and dimer(lane 2), but not rhVEGF(lane 4 and 5).
The result showed that specific antibodies against P277
were induced in mice immunized with rAnsB-TTP-P277.
Fig.3　Anti-P277 antibodies in immunized mice
Anti-P277 antibodies at a dilution of 1∶100 were detected by ELISA at several time points.A450 value of the
reaction solution was indicated with ordinate , and the age of mice were indicated with abscissa.Immunization
with chimeric enzyme of AnsB-TTP-P277 triggered stronger immune responses than with peptide P277 as shown
by the higher antibody levels.(n=10 mice per group)
734 Chinese Journal of Biochemistry and Molecular Biology Vol.23
Fig.4　Specificity of anti-P277 antibodies detected by Western blot analysis
(A)Proteins on nitrocellulose membrane stained by ponceau after being transferred from 15% SDS-PAGE gel.
(B)Result of Western blotting.
Lane1 , protein markers;lane 2 , rhVEGF-P277 with DTT;lane 3 , rhVEGF-P277 without DTT;lane 4 , rhVEGF
with DTT;lane 5, rhVEGF without DTT.To determine the specificity of anti-P277 antibodies produced in the
immunized mice , serum samples were tested by Western blot assay using rhVEGF-P277 fusion protein as antigen.
Antibodies from mice immunized with the chimeric enzyme of AnsB-TTP-P277 reacted with rhVEGF-P277 fusion
protein(lanes 2 and 3)but not with rhVEGF(lanes 4 and5).Under reducing conditions a single P277-reactive band
was observed to be compatible with the size of the rhVEGF-P277 monomer (lane 2), while under-non-reducing
conditions two P277-reactive bands were observed(lane 3)to indicate the formation of homodimers of rhVEGF-P277
2.3　rAnsB-TTP-P277 could promote the efficacy of
the peptide P277 itself on autoimmune diabetes
prevention in NOD mice
We immunized NOD female mice three times with
rAnsB-TTP-P277 in IFA at 4 weeks , 7 weeks and 10
weeks of age respectively.Control mice treated with PBS
in IFA for spontaneous IDDM appearing (Table 1 , group
A).In control group seven of the mice eventually
developed IDDM and only three escaped the disease , but
only one of the mice of group D eventually developed
IDDM and nine escaped the disease.The rAnsB-TTP-
P277 could reduce significantly the severity of
　　　　　
spontaneous diabetes developing (t-test , P <0.01).At
the age of 8 months the diabetic mice treated with the
chimeric L-asparaginase in IFA developed blood glucose
concentration of 6.2 mmol L compared to 22.5mmol L in
the control mice (Table 1 , group D).
Administration of the peptide P277 itself in IFA(Table 1 , group C) resisted the development of
spontaneous IDDM appearing , both incidence and severity
of IDDM were significantly reduced(t-test , P<0.01).
At age of 8 months there were 3 of 10 mice developed
IDDM of group C compared to 7 of 10 in the control mice.
Incidence of IDDM and blood glucose concentration of
　　
Table 1　Effect of administering antigens on development of diabetes
Groups Antigens
6 months 7 months 8 months
Incidence
Blood glucose
(mmol L) Incidence
Blood glucose
(mmol L) Incidence
Blood glucose
(mmol L)
A PBS 4 10 13.9±13.5 5 10 20.5±14.8 7 10 22.5±14.4
B AnsB 2 10 11.8±16.2 3 10 13.8±15.3 4 10 18.9±15.6
C P277 2 10 12.8±16.3 3 10 13.6±14.8 3 10 13.6±14.9
D RAnsB-TTP-P277 0 10 4.9±1.9 1 10 7.2±2.5 1 10 6.2±3.1
Four-week-old NOD female mice were treated by i.p.inoculationwith PBS or with 50μg of the indicated antigens in IFA.The mice were inoculated other twice
at 7 weeks and 10 weeks of age respectively.Spontaneous IDDM was assayed at ages 6 , 7 and 8 months as hyperglycemia(blood glucose ≥11.1 mmol L)
735No.9 ZHU Ai-Hua et al:L-asparaginase display of P277 for autoimmune diabetes prevention 　
Fig.5　Histological observation of pancreas
Pancreas tissue:sections stained with haematoxylin and eosin from control animals(A), from those immunization with
AnsB(B), P277(C)and rAnsB-TTP-P277(D).InA , the pathological section of pancreas islets tissue in PBS group,
HE×200.Many necrosis and marked at rophy ( ～ )of pancreas i slet s showed and many lymphocytes f iltrated
around the islets.In B , C and D , the pathological section of islet tissue in immunogenic group.A few necrosis areas
(+ ～ )formed in the pancreas tissue and a few lymphocytes fi ltrated around the islets of pancreas
group D and group C showed that rAnsB-TTP-P277 could
promote efficacy of prevention the diabetes by the peptide
P277 itself (t-test , P < 0.01). Furthermore
histochemical analysis of mice pancreas tissue showed that
rAnsB-TTP-P277 could decrease insulin and atrophy of
pancreas islets in NOD mice more efficiently than P277(Fig.5).
Group B in table 1 shows the effect of treating NOD
mice with L-asparaginase in IFA.The L-asparaginase
could protect the mice against spontaneous development of
IDDM too.Only 4 mice became diabetic compared to 7 of
10 mice given PBS in IFA alone at 8 months of ages.
3　Discussion
Our results indicated that displaying P277 on surface
of L-asparaginase could promote efficacy of autoimmune
diabetes prevention in NODmice.Studies showed that the
peptide P277 , an immunomodulatory peptide from hsp60 ,
could arrest β-cell destruction and maintain insulin
production in newborn diabetic NOD mice and in infant
patients (<6 months)with type I diabetes[ 6 , 7 ,9 , 19] .In
this paper , we have demonstrated a strategy of using a
fusion protein to enhance the antigenicity of P277 , which
is a weak antigen by itself.
The weak antigenicity of a auto-antigen can be
overcome by several methods , such as linking the auto-
antigen with strong T-helper epitopes like tetanus
toxin.[ 10] , or anchoring the auto-antigen on carrier
molecules like mycobacterial heat shock protein-65[ 20] , et
al.Here , asparaginase was chosen as carrier molecule ,
and tetanus toxin peptide was picked as the T-helper
epitope.Asparaginase(AnsB)can easily formed polymers
in solution.It had been used for the clinical application
in the treatment of leukemia and lymphosarcoma
[ 21 , 22] ,
and proved to be safe for future human vaccine
development.Once the P277 epitope was displayed on the
surface of each unit of enzyme multitmer , multiple P277
epitopes could be presented to APCs , such as
macrophages and dendritic cells which ingest large
antigens more effectively.Therefore , the high molecular
weight chimeric enzyme multimers would trigger strong
anti-P277 immunoreactions in vivo.
Single asparaginase molecule does not exhibit enzyme
activity until re-associated into multi-unit complexes[ 11] .
The chimeric enzyme of AnsB-TTP-P277 tetramers
maintained 83%of catalytic activity compared to their
native forms , indicating the hydrophobic P277 epitopes
did not interfere with the folding and assembling of the
proteins.The analysis of the published crystal structure of
asparaginase showed that its C-terminus was located in a
depression on the surface and faced to its interior
[ 21] .The
hydrophilic TTP spacer fused to the C-terminus of
asparaginase would presumably extend to the exterior of
the enzyme , thereby prevent the steric hindrance by
additional fused hydrophobic linear peptide , like P277 in
our case , and maintain the proper conformation of the
enzyme Moreover , TTP could also serve as a strong T-
helper epitope for triggering strong immune responses in
vivo after the chimeric enzyme was administrated to mice.
The tripeptide linker Ser-Gly-Thr allowed the flexibility in
the structure of the P277 epitope fused with TTP.
The P277 peptide can be exploited to regulate the
spontaneous autoimmune process of beta cell destruction.
P277 treatment was associated with the modulation of the
autoimmune reaction from a pro-inflammatory Th1 type to
an anti-inflammatory Th2 type.Investigation of the effects
736 Chinese Journal of Biochemistry and Molecular Biology Vol.23
of P277 in the NOD mouse model showed that the
modulation of the hsp60 autoimmunity was marked by a
down-regulation of T-cell autoimmunity to various auto-
antigens , such as insulin and glutamic acid
decarboxylase
[ 9] .The chimeric asparaginase rAnsB-TTP-
P277 could enhance the function of this modulation ,
reduce the autoimmune of beta cell destruction , thus
significantly reduce the disease severity during
spontaneous diabetes development.
It was found that immunizing NOD mice with
asparaginase alone could prevent the development of
diabetes in NOD mice , which had not been reported
previously..We did not yet know whether L-asparaginase
contained an epitope that cross-reacti with the P277 , or
other epitopes contributed to diabetes prevention.
Attempts to treat diabetes with non-specific immuno-
modulators , such as steroids and azathioprine , had shown
inconclusive results
[ 23] .Ciclosporin , however , conserves
endogenous insulin secretion , and was proved to change
the natural course of the disease through immune
intervention
[ 25]
We speculate that some potent immune
responses are induced at the initiation of spontaneous
immune responses and influenced the following course.,
and lead to the observed protective effects of L-
asparaginase in the NOD mice.We speculate that one
could observe anti-diabetic effects not only with specific
anti-antigens like P277 , but possibly with irrelevant
antigens.However , specific anti-antigens may still be
favored in immunization therapies in terms of the
reliability and the protective duration.
In summary , we have demonstrated that strong
immune response against P277 was successfully induced
in rAnsB-TTP-P277 immunized mice;.high level of anti-
P277 antibodies was detected in rAnsB-TTP-P277
immunized mice by ELISA and Western blot analysis;
NOD mice vaccinated with rAnsB-TTP-P277 could
significantly reduce the incidence of spontaneous
IDDM
[ d11] ..Our study has validated an effective strategy
of vaccine development against human native proteins ,
and its potential applications for the treatment of human
auto-immune diseases..
参考文献(References)
[ 1 ] 　Castano L , Eisenbarth G S.Type-I diabetes:a chronic autoimmune
disease of human , mouse , and rat [ J] .Annu Rev Immunol , 1990 ,
8:647-679[ 2 ] 　Harri son L C.Islet cell autoantigens in insulin-dependent diabetes:
Pandora s box revisited [ J] .Immunol Today , 1992 , 13:348-352[ 3] 　Elias D , Tikochinski Y , Frankel G , et al.Regulation of NOD mouse
autoimmune diabetes by T cells that recognize a TCR CDR3 peptide
[ J] .Int Immunol , 1999 , 11(6):957-966[ 4 ] 　Nussbaum G , Zanin-Zhorov A , Quintana F , et al.Peptide p277 of
HSP60 signals T cells:inhibi tion of inflammatory chemotaxis [ J] .Int
Immunol , 2006, 18(10):1413-1419[ 5 ] 　Birk O S , Elias D , Weiss A S , et al.NOD mouse diabetes:the
ubiquitous mouse Hsp60 is a beta-cell target antigen of autoimmune T
cells [ J] .J Autoimmun , 1996 , 9:159-166
[ 6 ] 　 Brugman S , Klatter F A , Visser J , et al.Neonatal oral
administration of DiaPep277 , combined with hydrolysed casein diet ,
protects against type 1 diabetes in BB-DP rats [ J] .Diabetologia ,
2004 , 47(7):1331-1333[ 7 ] 　Elias D , Cohen I R.Treatment or autoimmune diabetes and insulin is
in NOD mice with heat shock protein 60 peptide P277 [ J] .Diabetes ,
1995 , 44(9):1132-1138[ 8 ] 　Ablamunits V , Elias D , Reshef T , et al.Islet T cells secreting IFN-
gamma in NOD mouse diabetes:arrest by P277 peptide t reatment[ J] .J Autoimmun , 1998 , 11(1):73-81[ 9 ] 　Sia C.Imbalance in the cell polarization and its relevance in type 1
diabetes mellitus [ J] .Rev Diabetes Stud , 2005 , 2(4):182-186
[ 10] 　Panina-Borgignon P , Tan A , Termijtelen A , et al.Universally
immunogenic T cell epitopes promiscuous binding to human MHC
class II and promiscuous recognition by T cells [ J] .Eur J Immunol ,
1989 , 19:2237-2242[ 11] 　Marlborough D I , Miller D S , Cammack K A.Comparative study on
conformational stability and subunit interactions of two bacterial
asparaginases [ J] .Biochim Biophys Acta , 1975 , 386:576-589[ 12] 　Jingjing L , Xue Y , Agarwal N , et al.Human Muller cells express
VEGF183 , a novel spliced variant of vascular endothelial growth
factor [ J] .Invest Ophthalmol Vis Sci , 1999 , 40(3):752-759[ 13] 　Yankai Z , Taiming L , Jingjing L.Low temperature and glucose
enhanced T7 RNA polymerase-based plasmid stability for increasing
expression of glucagons-like peptide-2 in Escherichia coli [ J ] .
Protein Expr Purif , 2003 , 29:132-139
[ 14] 　Qi G F , Lin J , Cao R Y , et al.Asparaginase display of polypeptides
in the periplasm of Escherichia coli: potential rapid pepscan
technique for antigen epitope mapping [ J] .J Immunol Methods ,
2005 , 299:9-19[ 15] 　 Jinshu X , Jingjing L , Duan P , et al.The immunogenicity of
recombinant and dimeric gonadotrophin-releasing hormone vaccines
incorporat ing a T-helper epitope and GnRH or repeated GnRH uni ts[ J] .J Immunol Methods , 2004 , 289:111-112[ 16] 　 Songshan T , Zhenglan C , Jingjing L.Production and enhanced
biological activity of a movel GHRH analog , hGHRH with an N-
terminal Pro-Pro extension [ J] .Protein Expr Purif , 2004 , 34:296-
301
[ 17] 　Songshan T , Juanhui Z , Minghua D , et al.Construction and activity
of a novel GHRH analog , Pro-Pro-hGHRH(1-44)-Gly-Gly-Cys [ J] .
Acta Pharmacol Sin , 2004 , 25(11):1464-1470
[ 18] 　Wriston J C.Asparaginase [ J] .Methods Enzymol , 1970, 17:732-
742[ 19] 　Itamar R , Dana E , AnnA , et al.β-cell function in new-onset type 1
diabetes and immunomodulation with a heat-shock protein peptide(DiaPeP277):a randomized, double-blind , phase II t rial [ J] .
Lancet , 2001 , 358:1749-1753
[ 20] 　Perraut R , Lussow A R , Gavoille S , et al.Successful primate
immunization with peptides conjugated to purified protein derivative or
mycobacterial heat shock proteins in the absence of adjuvants [ J] .
Clin Exp Immunol , 1993 , 93:382-386[ 21] 　Swain A L , Jaskolski M , Housset D , et al.Crystal structure of
Escherichia coli L-asparaginase , an enzyme used in cancer therapy[ J] .Proc Natl.Acad Sci USA , 1993 , 90:1474-1478[ 22] 　Lubkowski J , Palm G J , Gilli land G L , et al.Crystal st ructure and
amino acid sequence of Wolinella succinogenes L-asparaginase [ J] .
Eur J Biochem , 1996 , 241:201-207[ 23] 　Achenbach P , FuchtenbuschM.Modulating the autoimmune response
in type 1 diabetes:a report on the 64th scientific sessions of the
ADA , June 2004 , Orlando , FL , USA [ J] .Rev Diabetes Stud ,
2004 Fall , 1(3):137-140
[ 24] 　Christie M R , Molvig J , Hawkes C J , et al.IA-2 antibody-negative
status predict s remission and recovery of C-peptide levels in type 1
diabetic patients treated with cyclosporin [ J] .Diabetes Care , 2002 ,
25(7):1192-1197
737No.9 ZHU Ai-Hua et al:L-asparaginase display of P277 for autoimmune diabetes prevention