An antigenic recombinant serine protease from Trichinella spiralis induces protective immunity in BALB/c mice.

In this study, we report the cloning and characterization of a cDNA encoding a Trichinella serine protease gene (TspSP-1.3) from GenBank. The recombinant TspSP-1.3 protein (rTspSP-1.3) was expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR analysis revealed that TspSP-1.3 was expressed at significantly higher levels in muscle larvae and adult worms than in newborn larvae. TspSP-1.3 was detected in excretory-secretory proteins of Trichinella spiralis with western blotting. Immunization with the rTspSP-1.3 antigen induced humoral immune responses, which manifested as elevated specific anti-rTspSP-1.3 IgG and IgE antibodies and a mixed Th1/Th2 response. To determine whether purified rTspSP-1.3 had good antigenicity and could be a vaccine candidate for the control of T. spiralis infection, we immunized BALB/c mice with rTspSP-1.3 and subsequently challenged the mice with T. spiralis larvae. The results showed that mice vaccinated with rTspSP-1.3 exhibited an average reduction in the muscle larvae burden of 39 % relative to the control group. These results suggest that TspSP-1.3 could be a novel vaccine candidate for controlling Trichinella infection.

Analysis of a novel cathepsin B circulating antigen and its response to drug treatment in Trichinella-infected mice.

In this paper, we cloned a novel full-length cDNA that encodes a Trichinella spiralis cathepsin B-like protease gene (TsCPB) using 3'-RACE PCR. The recombinant mature TsCPB protein (rTsCPB) was then expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR revealed that TsCPB was expressed across all development stages of the parasite but had the highest expression level during the adult stage. Furthermore, rTsCPB was detected in Trichinella excretory-secretory products with anti-rTsCPB rabbit polyclonal antibodies. Interestingly, rTsCPB was strongly recognized by the T. spiralis-infected sera in Western blotting, implying that TsCPB protein appeared in the peripheral blood of Trichinella-infected mice as circulating antigens (CAg). We then analyzed the dynamic levels of TsCPB CAg and its antibodies in T. spiralis-infected sera by using an improved double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA, respectively. The results showed that TsCPB CAg can be detected much earlier compared to antibody detection in Trichinella-infected mice. In addition, we monitored the effects of albendazole drug therapy (a dosage of 370 mg/kg body weight, twice a day) on T. spiralis-infected mice by detecting the levels of TsCPB CAg and its antibody in the sera of drug-treated mice. The results showed that the levels of CAg dramatically decreased after successful drug treatment, while the antibody level remained unchanged. Overall, the novel Trichinella antigen TsCPB could be a promising novel circulating antigen molecule for the detection of Trichinella infection and for monitoring the efficacy of drug treatment of trichinellosis.

[Expression and effects of microRNA-155 in the livers of septic mice].

OBJECTIVE: To evaluate the effects of microRNA-155 (miR-155) on liver injury in mice with sepsis. METHODS: One hundred and twenty BALB/c mice were randomly divided into two groups of equal number according to random number table. Sepsis was induced by intraperitoneal injection of lipopolysaccharide (LPS,20 mg/kg). The mice were sacrificed at the time-points of 0, 2, 6, 12, 24, 48 hours. Blood and liver tissue were collected, and the levels of tumor necrosis factor- α (TNF- α ), interleukin (IL-6, IL-10) in serum and liver homogenate and alanine transaminase (ALT) in serum were determined by enzyme linked immunosorbent assay(ELISA). The injury of liver tissue was evaluated by histopathology. The expression of miR-155 in liver tissue was assessed by fluorescent quantitation reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The levels of TNF- α , IL-6 and IL-10 in serum and liver homogenate of septic mice increased with passage of time, and then the levels of TNF-α and IL-6 lowered after reaching the peak value, but remained higher than that of control group.TNF-α (ng/L) reached the peak value at 2 hours post-LPS-injection (serum: 1538.46 ± 102.12 vs. 64.52 ± 18.44,liver homogenate: 255.26 ± 41.23 vs. 60.21 + 13.55, both P<0.05). The level of IL-6 (µg/L) reached the peak value at 6 hours post-LPS-injection (serum: 875.33 ± 102.37 vs. 153.72 ± 20.67, liver homogenate: 9.22 + 0.82 vs. 3.35 ±0.36, both P<0.05), and that of IL-10 (ng/L) reached the peak value at 48 hours post-LPS-injection (serum: 520.13 ± 88.52 vs. 23.43 3.01, liver homogenate: 260.12 + 50.38 vs. 16.37 ± 3.71, both P<0.05). There were significant differences in above indexes between septic and control group (all P<0.05). The serum level of ALT (U/L) rose with passage of time, reaching the peak value at 48 hours post-LPS-injection (603.26 + 70.21 vs. 45.84 + 5.64, P<0.05). The values showed significant differences between septic and control group (P<0.05). A large number of leucocytic infiltration was found in liver. Hepatic tissue showed architectural distortion. Hepatocyte vacuolation and nodular necrosis were obvious at 12 hours post-LPS-injection. Relative expression of miR-155 was found to be increased at 2 hours post-LPS-injection, reaching its peak value at 12 hours post-LPS-injection [(72.96 ± 9.34)-fold of control group, P<0.05]. CONCLUSION: The increase in miR-155 expression might play an important role in the mechanism of liver injury during sepsis.

[The protective effect of chronic schistosoma japonica infestation against sepsis in mice].

OBJECTIVE: To preliminarily study the protective effect of chronic schistosoma japonica (SJ) infestation against sepsis in mice and its mechanism. METHODS: BALB/c male mice were used, and the experiment was divided into three parts. Experiment 1: chronic SJ infestation model was reproduced by SJ cercaria inoculation through abdominal skin for 8 weeks. Twenty mice were randomly grouped into normal group (n=10) and SJ group (n=10). The levels of interleukins (IL-4, IL-10),tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum were detected by enzyme linked immunosorbent assay (ELISA). Real-time polymerase chain reaction (PCR) was employed to detect the levels of IL-10 mRNA and TNF-αmRNA in abdominal macrophages. This experiment was meant to evaluate immune state in mice with chronic SJ infestation. Experiment 2: lipopolysaccharide (LPS) was intraperitoneally injected to reproduce sepsis model. Thirty mice were randomly grouped into LPS group (n=15) and SJ-LPS group (n=15). The levels of cytokines were determined by ELISA at 0, 24, 48 and 72 hours after LPS injection. This experiment was meant to detect the effect of chronic SJ infestation in mice during the septic process. Experiment 3: two types of sepsis model were reproduced by cecal ligation and puncture (CLP) and LPS injection, respectively. The survival rate of mice with chronic SJ infestation in 72 hours in either type of sepsis was evaluated. RESULTS: Experiment 1: compared with normal group [IL-4 (56.32±8.66) ng/L, IL-10 (48.17±7.23) ng/L], chronic SJ infestation showed an increase in serum IL-4 [(151.35±12.24) ng/L] and IL-10 [(133.22±11.09) ng/L, both P<0.05]. Chronic SJ infestation also resulted in an increase in IL-10 mRNA expression (SJ group 4.46±1.82, normal group 1.52±0.60) and inhibited TNF-α mRNA expression (SJ group 1.61±0.93, normal group 2.32±1.03) in abdominal macrophages (both P<0.05), indicating that macrophages could be differentiated into alternative activated macrophages. Experiments 2 and 3 showed that the levels of serum IL-4 and IL-10 were increased at 0 hour after LPS injection, and then gradually decreased in SJ-LPS group, but the levels were still higher than those in LPS group at 72 hours [IL-4 (ng/L): 92.2±7.6 vs. 41.5±4.5; IL-10 (ng/L): 92.1±7.8 vs. 35.6±4.0, both P<0.05]; the levels of TNF-α and IFN-γ were increased at 24 hours, and then decreased in SJ-LPS group, and the levels were lower than those in LPS group at 72 hours [TNF-α (ng/L): 82.9±5.6 vs. 91.5±5.2; IFN-γ (ng/L): 44.1±4.8 vs. 52.6±4.0, both P<0.05]. Therefore, chronic SJ infestation could improve the survival rate of mice with sepsis induced by CLP or LPS (CLP: 80% vs. 20%, LPS: 70% vs. 30%, both P<0.05). CONCLUSION: Chronic SJ infestation could elevate anti-inflammatory factors in septic mice, thus ameliorating the survival rate, so it has protective effect on mice with sepsis.

[Preparation of polyclonal antibody of recombinant human thioredoxin-1 and its protective effects on neonatal rats with endotoxemia].

OBJECTIVE: To clone the gene human thioredoxin 1 (hTrx-1) expressing its protein in the E.coli expression system and to obtain its polyclonal antibody, and to study the protective effects of hTrx-1 on neonatal rats with endotoxemia. METHODS: DNA encoding hTrx-1 from fetal liver cells was isolated by RT-PCR. The hTrx-1 was cloned to the prokaryotic expression plasmid PET-28a to induce its protein expression in the E.coli expression system. The purified hTrx-1 was injected into rats to prepare polyclonal antibody. Newborn Sprague-Dawley rats were randomly assigned to three groups: control, lipopolysaccharide (LPS) and hTrx-1 (n=12 each). The control and the LPS groups were intraperitoneally injected with normal saline and LPS (5 mg/kg), respectively. The hTrx-1 group received an intraperitoneal injection of hTrx-1 (10 mg/kg) 30 minutes before LPS injection. The mortality rate 24 hrs after injection was compared between the three groups. RESULTS: The prokaryotic expression plasmid PET-28a-hTrx-1 was constructed. The hTrx-1 protein was expressed and purified. The polyclonal antibody of hTrx-1 with the titer of 1∶51200 was prepared. The mortality rate of the control, LPS and hTrx-1 groups was 0, 67% and 17%, respectively (χ2=14.400, P<0.01). CONCLUSIONS: The polyclonal antibody of hTrx-1 is prepared successfully. The hTrx-1 protein has protective effects on neonatal rats with endotoxiamia.

[Polymorphism and structural analysis of suppressor of cytokine signaling-1 of rat].

OBJECTIVE: To identify the suppressor of cytokine signaling-1 (SOCS-1) of rat from the amplified gene with the help of bioinformatics to predict the deduced protein's structure and function in order to lay the foundation for further theoretical study. METHODS: The full-length rat SOCS-1 gene was amplified and identified from the GeneBank Nucleotide database, and the corresponding structure and function of its deduced protein was predicted by the bioinformatics analyzing tools online and the complicated bioinformatics software package Vector NTI suite 8.0, meanwhile the molecular cladogram was reconstructed. RESULTS: Two sequences were obtained by polymerase chain reaction (PCR) amplification of different SOCS-1 gene. The gene was comprised of 639 base pairs in the length, deduced 212 amino acids (aa), contained a SOCS box (aa172-aa208), a SH2 domain (aa80-aa155) and a nuclear localization sequence (aa160-aa174). The primary structure contained two linear B cell epitopes, all of them were on the surface of the protein and far away from the spatial structure. CONCLUSION: SOCS-1 gene has a polymorphism, its conservative SH2 region and SOCS box are related to its inhibitory effect on the signal transduction pathway. The nucleic localization sequence may affect other nuclear transduction factors. The B cell linear epitopes may be a candidate of immunodiagnosis with promising prospects.

[Protective immunity of Cs-Rho GTPase recombinant protein against Clonorchis sinensis infection].

OBJECTIVE: To study the protective immunity induced by recombinant vaccination of Cs-Rho GTPase of Clonorchis sinensis (Cs). METHODS: 20 SD-rats(8 weeks) were divided into two groups: A (recombinant protein experiment group) and B (PBS control group). Rats in group A were immunized with 1 ml protein of Cs-Rho GTPase (90 microg/ml) and 1 ml Freund's complete adjuvant through back and vola. 2 week later, the rats were given 1 ml protein of Cs-Rho GTPase (90 microg/ml) and 1 ml Freund's incomplete adjuvant, followed by 1 ml protein of Cs-Rho GTPase (90 microg/ml) through intraperitoneal injection at 4, 7, 11 week after the first immunization. Rats in group B were given PBS in the same way as group A. All rats were challenged each with 50 Clonorchis sinensis metacercariae after the last immunization. 21 d later, fecal samples were collected from all rats for examining eggs (number of eggs per gram feces, EPG) in every 3-5 d. When eggs were found, the rats were sacrificed and worms were collected. IgG, IgG1 and IgG2a in sera were detected by ELISA before every immunization. Mean number of worms and eggs, and antibody level in the experiment group were calculated and statistically compared with the controls. RESULTS: The mean number of worms and EPG were (9.2 +/- 9.9) and (956.8 +/- 1 062.5) respectively in group A, which were significantly lower than those of group B [(23.25 +/- 15.75) and (3 062.5 +/- 2 501.8) respectively] (P < 0.05). The absorbency values of serum IgG (0.1, 0.45, 0.65, 0.6, 0.65), IgG1 (0.1, 0.45, 1.1, 1.0, 1.1), and IgG2a (0.1, 0.7, 1.1, 1.1, 1.1) before every immunization in group A were significantly higher than those of group B (almost always 0.1) (P < 0.05). CONCLUSION: Recombinant vaccination of Cs-Rho GTPase induces partial protective immunity against Clonorchis sinensis infection in rats.

[Expression and characterization of the FABP and Eg95 fusion gene from Echinococcus granulosus].

OBJECTIVE: To construct and express a fusion gene of fatty acid binding protein (FABP) with Eg95 which are protective antigen genes of Echinococcus granulosus, and investigate the immunological characteristics of the recombinant protein. METHOD: Using cDNA fragments encoding FABP and Eg95 genes from E. granulosus Qinghai sheep strain as templates, a fusion gene FABP.Eg95 was amplified by asymmetric polymerase chain reaction th-rough a linking sequence encoding four glycine residues. The PCR products of fusion gene were subcloned into the pET28a (+) vector. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells and induced with IPTG. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. Results The fusion gene length was about 795 bp. Double enzyme digestion analysis and DNA sequencing confirmed that the fusion gene was cloned into pET-28a (+). The recombinant protein (Mr 31,000) was expressed in inclusion body in E. coli expression system, and was purified by Ni-IDA affinity chromatography. Western blotting analysis testified that the recombinant protein could be recognized by sera of cystic echinococcosis patients, but not by sera of healthy persons and schistosomiasis patients. CONCLUSION: The FABP.Eg95 fusion gene has been constructed, and the purified recombinant protein has been confirmed with immunogenicity.

[Subcellular mimical localization of the ATP synthase B subunit from Clonorchis sinensis under different conditions of cell cycling].

OBJECTIVE: To investigate the subcellular localization of ATP synthase b subunit from Clonorchis sinensis under different conditions of Hela cell cycling, and the effect of this protein on the expression of its encoding-gene and homologous host genes. METHODS: pEGFP-N1-CsATP-synt_B and the vector pEGFP-N1 were transfected into Hela cells, respectively. Transfected cells were synchronized in G0/G1 by serum starvation, G1/S, S cells by double thymidine block, and G2/M cells by thymidine-Nocodozale block. After synchronization, the subcellular localization of the expressed fusion protein was observed with a laser confocal microscope. The expression level of this fusion protein in cells was detected by flow cytometry (FCM). The expression of CsATP-synt_B and HomoATP-synt_B in different cell cycle phases accessed by RT-PCR. RESULTS: FCM results indicated that in the G0/G1 phase the expression of pEGFP-N1 vector was decreased significantly, while pEGFP-N1-CsATP-synt_B expression showed an upward trend. In the other phases of cell cycle, the protein expression was similar in the above two kinds of plasmids. The intact CsATP-synt_B was expressed in mitochondria in the G0/G1, S, and G2/M phases and nucleus during G1/S phase. After the fusion proteins entered the nucleus, the mRNA expression of CsATP-synt_B and HomoATP-synt_B increased significantly. CONCLUSION: CsATP-synt_B can be expressed in the nucleus during G1/S phase, and regulated by the cell cycle and energy requirements.

[Experimental establishment of life cycle of Clonorchis sinensis].

OBJECTIVE: To establish and maintain the life cycle of Clonorchis sinensis in laboratory. METHODS: Adult worms and eggs of Clonorchis sinensis were collected from naturally infected cats. Eggs were ingested by freshwater snails in aquarium. When the cercariae were released from infected snails, they invaded into freshwater fishes. From the 30th day on after the release of cercariae, the infection rate and metacercariae density in freshwater fishes were determined. RESULTS: After 95 days the infected snails began shedding cercariae in a temperature range of 24.3 -37.2 degrees C, and no cercariae were found under 20 degrees C. The infection rate in the snails Parafossarulus striatulus and Alocinma longicornis was 12.5% and 18.0%, respectively. Metacercariae were found in fish at 30 days after cercariae infection, and matured metacercariae were detected in 45 days. The number of metacercariae per gram of fish meat in Pseudorasbora para, Ctenopharyngodon idellus, Rhodeus sinensis, Hypophthalmichthys nobilis, Cirrhinus molitorella, Carassius auratus, Cyprinus carpio and Oreochromis niloticus was 1 792, 16, 8, 6, 5, 4, 4, and 2, respectively. Rats and cats were fed with metacercariae from fish to receive adult worms. CONCLUSION: Life cycle of Clonorchis sinensis has been established and maintained in the laboratory.

[Tissular and subcellular localization of the ATP synthase B subunit in Clonorchis sinensis].

OBJECTIVE: To illustrate the distribution of ATP synthase b subunit in the tissue of Clonorchis sinensis adult and its subcellular mimical localization in HeLa cells. METHODS: With the antiserum against recombinant CsATP-synt_B protein raised from SD rats as primary antibody, paraffin sections of the adult of C. sinensis were processed by the method of fluorescent immunohistochemistry to observe the distribution of CsATP-synt_B protein in adult worm. According to the prediction by bioinformatics of the definite mitochondrial targeting sequence (MTS) and probable Bipartite nuclear localization signals (NLS_BP)in CsATP-syntB sequence, recombinant pEGFP-N1 plasmids containing the intact and three defective CsATP-synt_B sequence with single defect of MTS or NLS_BP or double defect respectively were constructed. The recombinant plasmids and the control plasmid-pEGFP-N1, pEYFP-Mito and H2B-CFP, were transfected into the HeLa cells by Lipofectamine 2000 reagent and the subcellular location of the GFP fusion protein was observed with confocal microscopy. RESULTS: The CsATP-synt_B protein appeared to distribute all over the adult worm, especially abundant on the acetabulum, ovary, vitellarium and tegument. The intact CsATP-synt_B was definitely expressed in mitochondria and/or nucleus of infected HeLa cells, whereas the MTS-deleted mutant only in cytoplasma and nucleus, the NLS_BP-deleted mutant in mitochondria and cytoplasm, and the double defect mutant only in cytoplasm. CONCLUSION: The distribution of CsATP-synt_B in adult is accord with that of mitochondria, and mainly exits in the organs and the tissues of active energy metabolism. This study first predicted and confirmed that CsATP-synt_B can be expressed in the nucleus.

[Prokaryotic expression, identification and histo-localization of the Taenia asiatica enolase gene].

OBJECTIVE: To express enolase gene of Taenia asiatica, investigate the immunoreactivity of the recombinant TaENO protein, and immuno-histo-localize the presence of the recombinant TaENO in adults of T. asiatica. METHODS: The gene encoding enolase of T. asiatica (TaENO) was cloned by high throughput sequencing from the cDNA library of adult T. asiatica. The coding region of TaENO was amplified by PCR, and cloned into a prokaryotic expression vector pET-30a (+). The recombinant plasmid was transformed into E. coli BL-21/DE3 and followed by expression of the protein induced by IPTG. The protein was purified by Ni-IDA affinity chromatography, and tested by SDS-PAGE. Its immunoreactivity was examined by Western blotting. The mice were immunized subcutaneously with purified TaENO formulated in Freund's adjuvant. Serum samples were collected and analyzed for specific antibodies by ELISA. The localization of TaENO in adult worms was demonstrated by immunofluorescent technique. RESULTS: The recombinant expression plasmid was identified by PCR, double endonuclease digestion and sequencing. The recombinant TaENO was about Mr 47 000 with a concentration of 0.37 mg/ml. It was recognized by antisera of SD rats immunized with TaENO, sera of taeniasis patients and sera of infected swine. The immunofluorescence assay revealed that TaENO immune serum located in the tegument of T. asiatica adult. CONCLUSION: The TaENO gene has been expressed with immunoreactivity, and the recombinant TaENO is immunolocalized in the tegument of T. asiatica adult.

[Informatics analysis of malate dehydrogenase from Taenia saginata asiatica].

Tools from bioinformatics websites such as NCBI, ExPaSy were used for the analysis. The malate dehydrogenase full-length gene from Taenia saginata asiatica was 1 212 bp in length, with a coding region of 30-1 028 bp and coding 332 amino acids. It was a complete and full-length gene compared with the homologues in GenBank. The protein showed no transmembrane region, with stable physical-chemical characteristics. Three major linear epitopes located aa95-aa100, aa322-aa327 and aa117-aa122, with certain distance from each other on the surface of spatial structure of malate dehydrogenase (MDH). The last one was the linear epitope of Taenia. This cytosolic malate dehydrogenase gene is a potential antigen for diagnosis.

[Cloning and prokaryotic expression of malate dehydrogenase gene of Taenia saginata asiatica and immunogenicity analysis of the recombinant protein].

OBJECTIVE: To clone and express the lactate dehydrogenase (LDH) gene of Taenia saginata asiatica and analyze the immunogenicity of the recombinant protein. METHODS: By screening the full length cDNA plasmid library, the coding region of LDH was amplified with PCR, and cloned into the prokaryotic expression vector pET-30a (+), then expressed in E. coli BL21 with IPTG induction. The recombinant protein was detected by SDS-PAGE and purified by Ni-IDA affinity chromatography, and its immunogenicity was analyzed by Western blotting. RESULTS: PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant expression plasmid was constructed. The expression products were obtained and purified by Ni-IDA affinity chromatography. Western blotting analysis of LDH recombinant protein testified that the recombinant protein could be recognized by sera of the Taenia saginata asiatica infected swine and the patient. CONCLUSIONS: The LDH gene of Taenia saginata asiatica has been cloned and expressed, and the purified protein has been confirmed with immunogenicity.

[Parasite-origin IgE-dependent histamine-releasing factors in inducing histamine release from sensitized mast cells].

OBJECTIVE: To obtain the recombinant IgE-dependent histamine-releasing factors of Schistosoma japonicum and Clonorchis sinensis (rSjHRF and rCsHRF) and to study the effect of recombinant HRFs to induce histamine release from sensitized rat mast cells. METHODS: The complete coding regions of SjHRF and CsHRF were cloned separately, and the recombinant plasmids were respectively transformed and expressed in BL21 cells. The soluble recombinant rSjHRF and rCsHRF were purified. Aliquots of the mast cells obtained from the lungs of OVA-immunized rats were separately incubated with rSjHRF and rCsHRF and the released histamine was measured by the OPT spectrofluorometric procedure. The dose-dependent curves and the kinetics of histamine release induced by rSjHRF and rCsHRF were prepared. RESULTS: The recombinant plasmids pET-30-rSjHRF and pET-30-rCsHRF were constructed successfully and the purified soluble recombinant proteins rSjHRF and rCsHRF were obtained by affinity chromatography. rSjHRF and rCsHRF induced histamine release from sensitized mast cells in a dose-dependent manner. At the concentration of 150 mg/L, the average rate of histamine release from sensitized mast cells induced by rSjHRF and rCsHRF were 49.78% and 32.63%, respectively. Histamine release increased with prolonged reaction time and the maximal release occurred at 35 min. CONCLUSION: The recombinant parasite-originated IgE-dependent HRFs show an effect of inducing histamine release from sensitized mast cells, suggesting that this protein would play a role in type I hypersensitivity in hosts with parasitic infections.

[Bioinformatics analysis for the structure and function of lactate dehydrogenase from Schistosoma japonicum].

OBJECTIVE: To predict the structure and function of SjLDH using bioinformatics method. METHODS: By online analysis at bioinformatics websites such as NCBI (http://www.ncbi.nlm.nih.gov/) and Expasy (http://cn.expasy. org/), and employing software packages such as Vector NTI suite and PCgene to do multi-sequence homological alignment, phylogenetic analysis, secondary structure and topological prediction, homology modeling of tertiary structure, antigenic epitope analysis, etc. RESULTS: Same conservative sites and key catalytic sites existed among SjLDH and LDHs from other species. Similarity of SjLDH compared to CsLDH, TvLDH and HsLDH was 75%, 17%, 58%-60% respectively. Phylogenetic analysis demonstrated that the evolution relation between SjLDH and DmLDH was closer than the relation between SjLDH and CeLDH, the relationship between SjLDH and HsLDH-B, -C was closer than HsLDH-A. Three transmembrane regions were found, the region of 98aa-106aa in three hydrophilic regions located outside of membrane was inferred as the major antigen epitope. This antigen epitope had significant difference with LDHs from protozoon (Pf., Tg., Tv.) and had 1-3 amino acid residue difference with MmLDH, HsLDH-A, -B, -C, and was the same with CsLDH. One of the key catalytic residues and substrate (pyruvate) binding loop were located in this region. Tertiary structure demonstrated that 98aa-106aa was on the surface of the protein and formed a substrate binding loop, other two key catalytic sites were at the position near the loop. CONCLUSION: The prediction implied that LDH was an ideal molecule for phylogenetic analysis; SjLDH might be a potential molecular target for immunodiagnosis, anti-schistosome drug and vaccine development.

[Cloning and expression of the F0-ATP synthase B chain of Clonorchis sinensis and immunogenicity identification of the recombinant protein].

OBJECTIVE: To clone and express the Clonorchis sinensis F0-ATP synthase b chain (CsF0-ATP-synt_B) gene and analyze immunogenicity of the recombinant protein. METHODS: The coding region F0-ATP synthase b chain gene with the mitochondrial targeting sequence (MTS) removed was amplified with PCR using the cloned plasmid as template, and the product was cloned into the prokaryotic expression vector pET-28a(+), transformed into E. coli BL21 (DE3) and induced with IPTG. The expressed product was purified by Ni-IDA affinity chromatography,and analyzed by SDS-PAGE for its expression and identified by Western blotting for its immunogenicity. RESULTS: The coding sequence of the F0-ATP synthase b-chain like gene removed off the MTS contains 813 base pairs encoding 271 amino acids with a theoretical molecular weight of 31,171.9. PCR, double enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-28a (+)-CsF0-ATP-synt_B was constructed successfully, and the resolvable expression was obtained in E.coli BL21. Highly purified recombinant protein was prepared through affinity chromatography. The recombinant protein could be recognized by the immune serum of the SD rat immunized with the recombinant protein. CONCLUSION: The CsF0-ATP-synt_B like gene has been efficiently expressed in prokaryotic expression system with immunogenicity.

[The effect of gossypol, praziquantel and artemether on recombinant lactate dehydrogenase from Schistosoma japonicum].

OBJECTIVE: To detect the effect of gossypol, praziquantel and artemether on activity of the recombinant lactate dehydrogenase of Schistosoma japonicum (rSjLDH). METHODS: Effect of gossypol (0-0.10 mmol/L), praziquantel (0-0.20mmol/L) and artemether (0-0.10 mmol/L) on the enzymatic activity of rSjLDH was detected by spectrophotometric method in standard reaction system established before, same solvent of each reagent was used as control. Results were analyzed by SPSS13.0 software. RESULTS: Compared with the control, considerable inhibition for both reduction and oxidation reactions catalyzed by rSjLDH was observed at 0.04 mmol/L of gossypol (P < 0.01). The enzyme activities of rSjLDH for reduction and oxidation reactions were inhibited by 91.3% and 89.1% respectively at 0.1 mmol/L of gossypol, com-pared with the control (P < 0.01). To praziquantel, enzymatic activity inhibition was observed at 0.02 mmol/L for oxidation reaction and at 0.06 mmol/L for reduction reaction (P < 0.05). At 0.2 mmol/L of praziquantel, enzymatic activities were inhibited by 83.8% for reduction reaction and 72.2% for oxidation reaction respectively, than that of the control (P < 0.01). No inhibition for both reduction and oxidation reactions was observed at 0.1 mmol/L of artemether, compared with that of the control (P > 0.05). CONCLUSION: Gossypol and praziquantel show a high inhibition on rSjLDH. SiLDH may be one of the molecular targets of praziquantel.

[Identification of the SjARG novel gene and study on its protective potential as a vaccine].

OBJECTIVE: To recognize and identify the arginase (ARG) gene of Schistosoma japonicum(Sj), and to study its protection potential as a vaccine. METHODS: The 5'-end of the ARG gene from the Sj cercariae cDNA library was amplified by nested-PCR and the sequence was identified by bioinformatics. The complete coding sequence(CDS) was cloned into pET30a (+) vector, and a recombinant SjARG protein (rSjARG) was expressed, purified and used to raise antibodies. ARG's activity as an enzyme was tested by ornithine-ninhydrin reaction. Western blotting was used to compare the immunologic characteristics of rSjARG with that of the native one in Sj adult worm. Indirect immunofluorescence assay was used to immunolocalize it. For evaluating the protection potential of rSjARG, mice were immunized by the recombinant protein and challenged by cercariae of S japonicum. RESULTS: The CDS length of the SjARG novel gene was identified as 1095bp rSjARG showed enzyme activity and the same immunologic characteristics with the native arginase in adult worm. SjARG located in the genital organ and gut of both sexes. The worm reduction rate and egg reduction rate in rSjARG group were 55.8% and 48.8% respectively, higher than that of the rSj26GST group (28.6% and 6.89% respectively). CONCLUSION: SjARG gene was identified, which shows a higher protection than the Sj26GST.

[Cloning and bio-informatics analysis of the CsCrSP from Clonorchis sinensis].

OBJECTIVE: To screen cDNA library from Clonorchis sinensis for gene identification, and to clone and construct library of secreted recombinant proteins. METHODS: cDNA library from Clonorchis sinensis was screened for identifying new genes by Blastn protocol, the sequence of which was further analyzed by Motifscan and NCBI Conserved Domain Search protocol. RESULTS: The secreted proteins (Pcs004f03) were identified, with a 689 bp DNA sequence (187aa), and a theoretical molecular weight of Mr 21100. It is predicted that the gene CsCrSP contains one N-glycosylation site, three N-myristoylation sites, three casein kinase II phosphorylation sites, one cAMP-and cGMP-dependent protein kinase phosphorylation site and protein kinase C phosphorylation site, a signal peptide at N-terminals and SCP-like extracellular protein domain. CONCLUSION: The CsCrSP gene is a secretory cysteine-rich protein with a SCP-like extracellular protein domain.