| Sparganosis is a serious zoonotic parasitic disease caused by infection with spargana, the plerocercoid larvae of some diphyllobothroid tapeworms belonging to the genus Spirometra, and it was mainly caused by spargana of Spirometra mansoni in China and southeast Asia countries. The clinical diagnosis of sparganosis is rather difficult and is often misdiagnosed because the larvae have no predilection for particular sites in the human body and the specific signs or symptoms are lacking. A definite diagnosis of subcutaneous sparganosis can be made by detection of the parasite in a biopsy tissue, but the confirmative diagnosis is very difficult for visceral and cerebral sparganosis since the larva is found only by surgical removal. ELISA using the crude or excretory-secretory (ES) antigens of plerocercoids has high sensitivity for detection of serum anti-sparganum antibodies in humans, but the main disadvantage is the cross-reactions with sera from patients with other parasitic diseases. In our previous studies, a sparganum cysteine protease (SeCP, ID:D63670; protein ID:BAA09820, GI:1834307) was identified from the crude and ES proteins of S. erinaceieuropaei sparganum. Cysteine protease (CP) is a kind of protein hydrolase which has cysteine residues in the active center of the enzyme and plays the principal roles in the development and survival of parasites and has been applied for diagnostic markers and vaccine targets for some parasitic diseases because of their immunogenicity. In this study, the full-length gene of SeCP was bioinformatics analysed, cloned and prokaryotic expressed. The recombinate SeCP was characterizd and evaluated of its potential application for serodiagnosis of sparganosis by detecting the specific antibody in the serum of mice and patients infected with spargana.Materials and methods1. sparganum, serum and experimental animalsThe spargana were obtained from subcutaneous tissues and muscles of naturally infected frogs, which were collected from Kaifeng Henan province, China. Test serum includes serum samples of mice infected with spargana, Trichinella sprialis, Toxoplasma gondii and Schistosoma japonicum, and serum of patients with sparganosis and other parasitosis. Cloning vector pGEM-T (Promega), Escherichia coli DH5α, prokaryotic expression vector pMAL-c2X and TB1 were all purchased from New England Biolabs.2. Cloning, expression and identification of SeCPThe structures and functions of SeCP were predicted using bioinformatical sonline sites and software programs (NCBI, EMBI, ExPasy, et al). The full length SeCP gene was cloned, expressed and recombinant SeCP (rSeCP) was purified. BALB/c mice were immunized with rSeCP, and the immune sera were collected and analyzed. The Western blotting was used to identifying the antigenicity and immunogenicity of rSeCP. The transcription, expression and location of the SeCP gene in different development stages were detected by RT-PCR and IFT, respectively.2. Enzyme activity identification and function analysis of rSeCPEnzymatic activity of rSeCP was assayed by using a substrate gel electrophoresis of 12% SDS-PAGE gel copolymerized with 0.1% gelatin as substrate and also was ietermined by cleavage of the fluorescent substrate Z-Phe-Arg-AMC (Sigma) as previously described. The fluorescence intensity was continuously measured with spectrophotofluorometry using excitation and emission wavelengths of 355nm and 160 nm. Enzyme activity affected by temperature, pH, metal ions (Cu, Mn, Zn) and he impact of specific inhibitors were determination. Rabbit immunoglobulin, human immune globulin, bovine hemoglobin (Hb), fibronectin, collagen I, collagen V and bovine serum albumin were chosen to determine the rSeCP hydrolysis function. rSeCP immunized serum with different dilutions (diluted at 1:50,1:100, :500, and 1:1000, respectively) were used to detected the serum inhibition to the enzyme activity. rSeCP incubation with E- 64 as a positive control. To determine he cytotoxic effects of anti-rSeCP antibodies to spargamum, an in vitro ADCC issay was performed.3. IgG antibodies detection in mouse sera infected with spargana and atient sera with trichinellosis using rSeCPUsing rSeCP and ES antigens of sparganum, we established the rSeCP-ELISA ind ES-ELISA to detected the IgG level in the mouse serum (0-28dpi) of heavily nfected group (5 spargana/mouse), moderate infected group (3 spargana/mouse), ind lightly infected group (1 sparganum/mouse) of spargana, respectively. Mouse erum of other parasites and patient’s sera with sparganosis and other parasitosis were also detected, and their sensitivity and specificity were evaluated.4. Statistical analysisAll statistical analyses of data were done with SPSS for Windows, version 17.0 SPSS Inc., Chicago, IL). Chi-square test and repeated measures of analysis of ariance (ANOVA) were used to determine the difference among the groups at arious periods. Intra-and intergroup statistical analyses were performed with one-way ANOVA (LSD test). The statistical significance was defined as P< 0.05.Results1. Cloning, expression and identification of SeCP geneBioinfermatics analysis indicated that the SeCP gene sequence was of 1 053 bp ength with a 1011 bp biggest ORF encoding 336-amino acid protein with a complete cathepsin propeptide inhibitor domain and a peptidase CIA conserved lomain and has a signal peptide site with had good antigenicity.The full length SeCP gene was cloned; recombinant SeCP (rSeCP) was expressed and purified. mmunuization of mice with rSeCP induced the Th2-predominant immune esponses.Western blotting showed rSeCP was recognized by serum of spargnum-infected mice, and anti-rSeCP serum recognized the native SeCP protein of spargnum crude or ES antigens. Expression of SeCP was observed at sparganum stages, but not at adult and egg. An immunolocalization identified SeCP in spargnum tegument and parenchymal tissue.2. Enzyme activity identification and function analysis of rSeCPThe protease activity of purified rSeCP was investigated by gelation SDS-PAGE assay. When the gels were incubated in an acidic buffer (pH5.5), a zone of hydrolysis was visible, and the successful inhibition of its enzyme activity with E-64, a specific cysteine proteinase inhibitor, demonstrated that the purified rSeCP was the expected cysteine protease. rSeCP showed a broad range of pH optima, from 3.5 to 8.5, and maximum activity was detected at pH5.5 and the optimum temperature was 37℃, Km=1.56 nM/min and Vm=4.051 nM/min. When added to the assay environment in the form of chlorides, the rSeCP catalytic activity was inhibited by irons (Cu++, Mn++ and Zn++) and E-64 with dose-dependent. rSeCP had function of degrading different proteins and the proteins were degraded more thoroughly along with the increase of the rSeCP amount. Evaluation of the inhibition of rSeCP catalytic activity by anti-rSeCP antibodies was determined using human Ig as the substrate and the result showed that the rSeCP activity inhibited by anti-rSeCP serum with dose-dependent. Anti-rSeCP antibodies had the potential capabilities of killing sparganum in ADCC assay.3. Antibody detection of mouse infected with sparganum by rSeCPIn heavily, moderately and lightly infected mice, anti-sparganum antibodies were firstly detected by rSeCP-ELISA at 10,10, and 12 dpi, respectively; the detection rate reached 100% at 14,22 and 22 dpi, respectively. While the anti-sparganum antibodies were firstly detected by ES-ELISA at 6,8 and 8 dpi, respectively; the detection rate reached 100% at 8,12 and 14 dpi, respectively. When rSeCP-ELISA was performed, anti-sparganum antibody levels in three groups of infected mice increased continually after infection, the seroconversion was observed in all groups of mice at 22 dpi and lasted to 28 dpi when the experiment was ended. The anti-sparganum antibody levels determined by both ELISA in three groups of infected mice were statistical different (FrseCP=10.029, FES=5.901, P< 0.05). Moreover, the serum antibody levels at different times after infection were also significantly different (FrSeCP=308.184, FES=601.727, P<0.05); there were interaction between detection time and infection level (FrseCP=4.266, FES=5.808, P <0.05).4. Sensitivity and specificity detection of rSeCPThe specific anti-sparganum IgG antibodies in serum samples of mice infected with sparganum and other parasites were determined and the results are shown that the detection rate of anti- sparganum antibodies in sparganum-infected mice was 96.67% by rSeCP-ELISA and 100% by ES-ELISA, respectively; the sensitivity of both ELISA was no significant difference (P>0.05). The specificities of both ELISA assays were 100% (77/77) as they did not show any false positive reaction with sera of mice infected with other parasites and non-infected normal mice. The sensitivity of rSeCP-ELISA and ES antigen ELISA was 100% when performed on sera of patients with spargarnosis; The specificity of rSeCP-ELISA and ES antigen ELISA was 98.22%(166/169) and 87.57%(148/169), respectively (P＜0.05).Conclusions1. Constructed the recombinant plasmid pMAL-c2X-SeCP/TB1 of SeCP and soluble rSeCP protein was induced and purified. The transcription and expression of SeCP gene was only detected in the sparganum but not in adult worms and eggs, mainly located in tegument and parenchymal tissue.2. rSeCP has high immunogenicity and antigenic protein, and possesses the natural protease activity and the function of hydrolysates the host proteins.3. rSeCP has good sensitivity to detect the specific IgG antibodies in mouse sera infected with spargana and patient sera with sparganosis, the specificity was significantly greater than that of ES, rSeCP could be uesd for serological diagnosis for sparganosis. |
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