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https://www.abbs.info ISSN 0582-9879 |
Molecular Cloning,
Expression and Vaccination of a Novel Gene Sj-MA of Schistosoma
japonicum
CHEN
Yu-Xiao, YI Xin-Yuan1*,
ZENG Xian-Fang1, YUAN Shi-Shan1, ZHANG Shun-Ke1,
MCREYNOLDS Larry2
(Department of Immunology,
Xiangya School of Medicine, Central South University, Changsha 410078, China;
1Department of Parasitology, Xiangya School of Medicine, Central
South University, Changsha 410078, China; 2Molecular Parasitology
Group, New England Biolabs, Inc., Beverly, MA01915, USA)
Abstract In order to obtain new gene and to develop the
new vaccine candidate of immunoprotection against Schistosoma japonicum (Sj),
Sj adult worm cDNA library was screened with anti-sera to soluble male
adult worm antigen and resulted in the discovery of a novel gene designated
as Sj-MA. Sequence analysis showed that Sj-MA as a complete cDNA
contains one open reading frame. It was deduced to contain 249 amino acid residues
and encode a 28.8 kD soluble protein with plenty of phosphorylation sites, supposing
its action in signal transduction. Furthermore, Sj-MA cDNA was cloned
into a prokaryotic expression vector pGEX-5X to construct the recombinant plasmid
which was transformed and highly expressed in E. coli as a 54.8 kD glutathione-S-transferase
(GST) fusion protein. The fusion protein rSj-MA/GST could be recognized
with both anti-male adult worm sera and anti-GST sera in Western blotting. Mice
vaccinated with the fusion protein revealed significant worm reduction rate
of 34.29% (P<0.001), compared with the control groups. Taken together,
the novel gene Sj-MA can be expressed in E.coli as a fusion protein
that can elicit immunity against Schistosoma japonicum, suggesting its
potential as a new vaccine candidate.
Key words
immunoscreening; Schistosoma japonicum;
male adult worm; expression
Screening cDNA library is an efficient
method for isolation of novel genes［1－3］. A lot of vaccine candidates for
schistosome are discovered by screening cDNA library of adult worm, schistosomulum,
cercaria and egg using immunized sera［4－7］. To explore new antigen, we for
the first time used sera against the soluble male worm antigen to screen Sj
adult worm cDNA library and characterized the expressed protein of the novel
gene. The study may provide evidence for research on new vaccine candidates
of Schistosoma japonicum.
1 Materials and Methods
1.1 Preparation of soluble Sj male adult worm antigen
The house rabbit was infected with 2000 Sj cercariae and forty-five
days after the infection it was sacrificed to recover adult worms by portal
vein perfusion. Then the male adult worms were selected and ground on ice
to prepare soluble male adult worm antigen.
1. 2 Preparation of sera
Three house rabbits were vaccinated by subcutaneous injection with 0.2 mg
of soluble male adult worm antigen prepared as above emulsified in FCA, FIA
(Freund’s complete adjuvant, Freund’s incomplete adjuvant from Sigma) of the
same volume for the primary and two boost vaccinations respectively. Two weeks
after last immunization, blood sample was collected by carotid artery and
sera were prepared with regular method.
1. 3 Immunoscreening of Sj
adult worm cDNA library
Sj adult worm cDNA library and E.coli SURE cell were gifts from Dr. McManus
of Queensland Institute of Medical Research, Australia. XPORT，XLOLR and Helper
phage 708 were products of Stratagene Co. Horseradish peroxidase conjugated
SPA was from New England Biolabs, Inc. Immunoscreening was performed according
to standard protocol. The sera used for immunoscreening were pre-absorbed
with E. coli SURE cell lysate. After three rounds of immunoscreening,
the positive phage clones were excised in vitro with helper phage 708 to obtain
their phagemids.
1. 4 DNA sequencing
After quick identification of the positive phagemids with PCR (T7 primer TAATACGAC-TCACTATAGGG
as the forward and T3 primer ATTAACCCTCAC TAAAGGGAA as the reverse) the PCR
product sequencing was performed by ABI PRISM 377XL DNA Sequencer. Sequence
with score less than 50 by Nucleotide BLAST of NCBI indicated to be a novel
gene and the deduced protein properties were analyzed with Expert Protein
Analysis System through Internet.
1. 5 Subcloning and prokaryotic expression
The novel gene designated as Sj-MA was PCR amplified with primers (TaKaRa
Co., China) flanked with BamHI and XhoI (New England Biolabs
Inc), 5′-CGGGATCCCGATGACTGGAGGGTCGGAAG-3′ as the forward and 5′-CCCTCGAGGCCGGAAACTT-GCCTGTTC-3′
as the reverse. The PCR condition was 94 ℃ 1 min, followed by 30 cycles of
94 ℃ 1 min 20 s, 60 ℃ 1 min 20 s and 72 ℃ 1 min 20 s, and finally 72 ℃ for
7 min. Double digested with BamHI and XhoI, both the PCR product
and the pGEX-5X vector proceeded to low melting temperature agarose electrophoresis
and the excised DNA bands treated with β-agarase I (New England Biolabs Inc.)
were ligated, then transformed into E.coli ER2688 for expression. The
E. coli with recombinant plasmid was grown at 37 ℃ with 100 mg/L ampicillin,
and isopropylthio-β-D-galactoside (IPTG) was added to a final concentration
of 1.0 mmol/L when optical density of the culture was 0.4. After growing for
another three hours, the cultured E. coli were harvested
by centrifuge at 4000 r/min for 15 min at 4 ℃. The pellet was suspended in
buffer A (50 mmol/L Tris・HCl, pH 8.0) and lysed by sonication on ice. After
spinning 10 000 r/min for 20 min at 4 ℃, the pellet resolved in buffer A containing
8 mol/L urea and centrifuged at 10 000 r/min for 10 min, the supernatant containing
the fusion protein was stored at －20 ℃.
1. 6 Identification of the recombinant
protein
10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were
used to analyze size and antigenicity of rSj-MA/GST. Briefly, gels
after electrophoresis were Coomassie blue-stained to observe protein bands
or transferred onto nitrocellulose membranes for Western blot using rabbit
sera to Sj male adult worm antigen and goat sera to Sj GST (Amersham
Biosciences) as the first antibody respectively. The second antibodies (Amersham
Biosciences) were HRP-conjugated goat anti-rabbit IgG and HRP-conjugated rabbit
anti-goat IgG. Then demonstrated with 0.05% diaminobenzoic acid.
1. 7 Immunoprotection assessment
Female Kunming mice were randomly divided into three groups. Before applied
to mice the fusion protein rSj-MA/GST was dialysed ［1 mol/L urea, 2
mmol/L reduced glutathione, 0.02 mmol/L oxidized glutathione, 0.005% Tween-80
and 0.05 mol/L Tris・HCl (pH 8.0)］ to discard urea followed by PBS (0.2 mol/L,
pH 7.2) for protein renaturing. The test group was subcutaneously injected
with 50 μg fusion protein emulsified in the same volume of FCA, while two
control groups with same dosage of GST(prepared in our lab) dissolved in PBS
plus FCA and the same volume of PBS respectively. Two weeks after the third
vaccination mice were challenged with (40±1) Sj cercariae while the
blood samples were collected for detection of specific antibody with enzyme-linked
immunosorbent assay (ELISA). Mice were sacrificed and perfused 6 weeks after
the challenge, and the immunoprotection was assessed by worm reduction percentage.
Statistics variance was analyzed with one-way ANOVA and P<0.05 indicated
significant difference.
2 Results
2.1 Isolation and identification of the novel gene Sj-MA
Eleven positive phage clones were isolated through immunoscreening the Sj
adult worm cDNA library after 3 rounds of screening. Among them, clone 2 designated
as Sj-MA proven to be a new gene by GenBank with accession number AF519808
contains one complete ORF with initiation codon ‘ATG’ at nucleotide site 17－19
and termination codon ‘TAA’ at nucleotide site 774－776 followed by a poly(A)
tail(Fig. 1).
Fig. 1 The nucleotide and deduced
amino acid sequences of Sj-MA
The initiation codon and termination codon are in italic
2.2 Deduced primary and secondary
structure
The putative amino acid sequence of the novel gene Sj-MA consisted
of 249 amino acid residues for a molecular mass of 28.8 kD protein predictably
as a soluble protein. For its secondary structure Sj-MA was predicted
both by SOPMA analysis to consist of 100% of α-helix and by Scanprosite analysis
to contain nine protein kinase C phosphorylation sites (amino acid 35－37,
54－56，68－70，88－90，91－93，123－125, 137－139, 227－229 and 234－236), three casein
kinase II phosphorylation sites (amino acid 20－23，76－79 and 84－87), two tyrosine
kinase phosphorylation sites (amino acid 121－127 and 224－232) and one N-myristoylation
site (amino acid 4－9).
2.3 Cloning, expression and identification
The cDNA fragment encoding Sj-MA was correctly in frame with the expression
vector pGEX-5X, which was confirmed with both double enzyme digestion and
with PCR. Expression of the GST(26 kD) fusion protein rSj-MA/GST was
visualized as 54.8 kD in large quantity in 10% SDS-PAGE (Fig.2) and existed
as inclusion body soluble in 8 mol/L urea. Antigenicity of rSj-MA/GST
was demonstrated in Western blot by the recognition with both sera against
male adult worm antigen and sera against GST, but not with normal sera (Fig.3,
4 ).
Fig. 2 Analysis of fusion protein
expression rSj-MA/GST with SDS-PAGE
M, protein marker; 1, expressed fusion protein (arrow) in 8 mol/L urea; 2,
supernatant of expressed E. coli lysate induced with IPTG; 3, control
E. coli lysate induced with IPTG in 8 mol/L urea; 4, control E.
coli lysate without IPTG induction.
Fig. 3 Analysis of rSj-MA/GST
with Western blot
M, protein marker; 1, 5, expressed fusion protein in 8 mol/L urea; 2, supernatant
of expressed E. coli lysate induced with IPTG; 3, control E.coli
lysate induced with IPTG in 8 mol/L urea; 4, expressed GST control induced
with IPTG in 8 mol/L urea. 1, reacted with normal goat sera； 2－5, reacted
with anti-GST goat sera.
Fig. 4 Antigenicity analysis
of rSj-MA/GST with Western blot
M, protein marker; 1,4, expressed rSj-MA/GST in 8 mol/L urea; 2, supernatant
of the expressed E. coli lysate induced with IPTG; 3, control E.
coli lysate induced with IPTG in 8 mol/L urea. 1－3, reacted with rabbit
sera to soluble male adult worm antigen; 4, reacted with normal rabbit sera.
2.4 Immunoprotection assessment
Vaccination with fusion protein rSj-MA plus FCA elicited specific IgG
with titer up to 1:12 800 in mice 2 weeks after the last vaccination, which
was significantly higher than the controls. Mice were sacrificed and perfused.
The recover worms were counted. Significant worm reduction rate (34.29%, P
< 0.001) was observed in the test group compared with the controls (Table
1).
Table 1 Worm reduction in mice
vaccinated with rSj-MA/GST
| Groups | Mice(n) | Worm recovered (x±s) | Worm reduction rate vs. PBS (%) |
| rSj-MA/GST+ FCA | 9 | 20.10±5.30* | 34.29 |
| GST+ FCA | 9 | 27.95±4.99# | 8.62 |
| PBS | 10 | 30.59±4.35 | – |
*P<0.001 vs. PBS, GST+FCA; #P> 0.05 vs. PBS.
3 Discussion
Many new genes and their proteins were obtained through screening cDNA library
of schistosome and subsequent prokaryotic expression in E.coli［8－10］.
Schistosomes are hermaphrodite. Pairing with male schistosomes is required
by females to achieve sexual maturity for pathogenesis in schistosomiasis.
We for the first time use anti-male adult worm antigen sera to screen Sj
adult worm cDNA library which might possibly obtain new gene of male worm
origin. Cloning and prokaryotic expression of the novel gene enable us to
obtain large amount of the encoded protein for immunological studies and evaluation
as a vaccine candidate. In this study, we for the first time report the cloning,
characterization and vaccination of a novel gene Sj-MA. Sequence analysis
showed that Sj-MA contained a complete cDNA with one open reading frame.
It was deduced to contain 249 amino acid residues and to encode a 28.8 kD
soluble protein with nine protein kinase C phosphorylation sites, three casein
kinase II phosphorylation sites, two tyrosine kinase phosphorylation sites
and one N-myristoylation site, indicating its possible action in signal transduction.
By cloning into pGEX-5X the novel gene was effectively expressed as GST fusion
protein rSj-MA/GST with proven antigenicity by Western blotting using
both anti-male adult worm sera and anti-GST sera. Mice vaccinated with rSj-MA/GST
elicited high level of specific antibody as well as 34.29% worm reduction
rate, demonstrating its potential as a new vaccine candidate against schistosomiasis.
Investigation in future on gender-specificity of Sj-MA and its action
against worm development is of great importance.
Acknowledgements We thank Dr. Don Comb (New England Biolabs, Inc. USA)
for his kind support to the project and professor DP McManus (Molecular Parasitology
Unit, Queensland Institute of Medical Research, Australia) for kindly presenting
Sj cDNA library to us.
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Received: July 2, 2003 Accepted:
August 18, 2003
This work was supported by a grant from WHO/TDR( No. 980268 )
*Corresponding author: Tel, 86-731-4498311; Fax, 86-731-4498311; e-mail, [email protected]
Updated at: 12-18-2003