Recombinant Toxoplasma gondii phosphoglycerate mutase 2 confers protective immunity against toxoplasmosis in BALB/c mice.

Toxoplasmosis is one of the most widespread zoonoses worldwide. It has a high incidence and can result in severe disease in humans and livestock. Effective vaccines are needed to limit and prevent infection with Toxoplasma gondii. In this study, we evaluated the immuno-protective efficacy of a recombinant Toxoplasma gondii phosphoglycerate mutase 2 (rTgPGAM 2) against T. gondii infection in BALB/c mice. We report that the mice nasally immunised with rTgPGAM 2 displayed significantly higher levels of special IgG antibodies against rTgPGAM 2 (including IgG1, IgG2a and IgAs) and cytokines (including IFN-γ, IL-2 and IL-4) in their blood sera and supernatant of cultured spleen cells compared to those of control animals. In addition, an increased number of spleen lymphocytes and enhanced lymphocyte proliferative responses were observed in the rTgPGAM 2-immunised mice. After chronic infection and lethal challenge with the highly virulent T. gondii RH strain by oral gavage, the survival time of the rTgPGAM 2-immunised mice was longer (P < 0.01) and the survival rate (70%) was higher compared with the control mice (P < 0.01). The reduction rate of brain and liver tachyzoites in rTgPGAM 2-vaccinated mice reached approximately 57% and 69% compared with those of the control mice (P < 0.01). These results suggest that rTgPGAM 2 can generate protective immunity against T. gondii infection in BALB/c mice and may be a promising antigen in the further development of an effective vaccine against T. gondii infection.

DNA vaccination with a gene encoding Toxoplasma gondii Rhoptry Protein 17 induces partial protective immunity against lethal challenge in mice.

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8(+) T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.

[Research Advances on Gliding-associated Proteins of Toxoplasma gondii].

The ability to invade host cells is a key to the survival and pathogenicity of Apicomplexan parasites. Toxoplasma gondii is an obligatory intracellular parasite. Its motility, invasion into, and egression from host cells are powered by a machinery called acto-myosin motor （AMM）. The AMM is composed of myosin A, a myosin light chain （MLC1）, two essential light chains （ELC）1, 2 and gliding-associated protein （GAP）. The GAP family has been discovered to include GAP45, GAP50, GAP80, GAP70 and GAP40, which are the major components of glideosome that provides power for parasite motility. The glideosome of apicomplexan parasites is an actin- and myosin-based power machine located at the pellicle between the plasma membrane （PM） and inner membrane complex (IMC). This review outlines our current understanding of GAP function and architecture as well as the molecular basis for parasite motility. Meanwhile, the use of GAPs as the candidate toxoplasmosis vaccine is prospected.

[Toxoplasma gondii Rhoptry Protein 17 Inhibits the Apoptosis of Mouse Macrophages via Activation of Activator Protein 1 Signaling].

Objective: To investigate the effect of Toxoplasma gondii rhoptry protein 17（ROP17） on γ-interferon (IFN-γ)-induced apoptosis of mouse J774A.1 monocyte macrophages. Methods: The J774A.1 cells were transfected with recombinant plasmid p3×Flag-CMV-14/TgROP17 or empty plasmid p3×Flag-CMV-14. After addition of IFN-γ, flow cytometry and Western blotting were performed to detect apoptosis and the protein levels of phosphorylated c-Jun and apoptosis-related proteins cleaved Caspase-3, Bcl-2, Bcl-xL and Bcl-3. The p3×Flag-CMV-14/TgROP17 plasmid and c-Jun shRNA were co-transfected into J774A.1 cells, after which IFN-γ was added to induce cell apoptosis. The levels of cleaved Caspase-3 and Bcl-3 were analyzed using Western blotting. Results: Flow cytometry showed that the apoptosis rate of cells overexpressing ROP17［（3.73±0.51）%ï¼½ was significantly lower than that of the control cells［（7.78±1.10）%, P<0.05ï¼½. Western blotting showed significant differences in protein levels of phosphorylated c-Jun（0.196±0.028 vs. 0.075±0.010）, Bcl-3（0.461±0.063 vs. 0.108±0.013） and cleaved Caspase 3（0.015±0.004 vs. 0.174±0.026） between the cells overexpressing ROP17 and control cells （all P<0.05）. However, the levels of Bcl-2 and Bcl-xL were not significantly different between the cells overexpressing ROP17 and the control. When the expression of c-Jun and phosphorylation of c-Jun were inhibited by c-Jun shRNA, the relative level of cleaved Caspase 3 in the RNA interferenced cells and control cells was 0.147±0.024 and 0.087±0.010, respectively (P<0.05), and the relative level of Bcl-3 was 0.085±0.010 and 0.162±0.011, respectively (P<0.05). Conclusion: The anti-apoptosis effect of ROP17 is dependent on the phosphorylation of c-Jun and the expression of Bcl-3.

[Research Advance on Toxoplasma gondii Rhoptry Protein 2 Family as Candidate Vaccine Molecules].

Toxoplasma gondii rhoptry protein 2 family (ROP2 family), secreted by the rhoptry, plays an important role in T. gondii invasion of host cells and its virulence. The ROP2 family members include ROP2, ROP4, ROP5, ROP8, ROP13, ROP16, ROP17, and ROP18. Recent studies have found that these members are potential vaccine candidates against toxoplasmosis, and can induce the protective immunity of the host. This paper reviews the research advance on the ROP2 family members as DNA or protein vaccines against toxoplasmosis.

Protective immunity induced by peptides of AMA1, RON2 and RON4 containing T-and B-cell epitopes via an intranasal route against toxoplasmosis in mice.

BACKGROUND: Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis. Apical membrane antigen-1 (AMA1) and rhoptry neck protein (RON2, RON4) are involved in the invasion of T. gondii. METHODS: This study chemically synthesized peptides of TgAMA1, TgRON2 and TgRON4 that contained the T- and B-cell epitopes predicted by bioinformatics analysis. We evaluated the systemic response by proliferation, cytokine and antibody measurements as well as the mucosal response by examining the levels of antigen-specific secretory IgA (SIgA) in the nasal, vesical and intestinal washes obtained from mice after nasal immunization with single (AMA1, RON2, RON4) or mixtures of peptides (A1 + R2, A1 + R4, R2 + R4, A1 + R2 + R4). We also assessed the parasite burdens in the liver and brain as well as the survival of mice challenged with a virulent strain. RESULTS: The results showed that the mice immunized with single or mixed peptides produced effective mucosal and systemic immune responses with a high level of specific antibody responses, a strong lymphoproliferative response and significant levels of gamma interferon (IFN-γ), interleukin-2 (IL-2) and IL-4 production. These mice also elicited partial protection against acute and chronic T. gondii infection. Moreover, our study indicated that mixtures of peptides, especially the A1 + R2 mixture, were more powerful and efficient than any other single peptides. CONCLUSIONS: These results demonstrated that intranasal immunisation with peptides of AMA1, RON2 and RON4 containing T- and B-cell epitopes can partly protect mice against toxoplasmosis, and a combination of peptides as a mucosal vaccine strategy is essential for future Toxoplasma vaccine development.

Partial protective effect of intranasal immunization with recombinant Toxoplasma gondii rhoptry protein 17 against toxoplasmosis in mice.

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.

Intranasal immunisation of the recombinant Toxoplasma gondii receptor for activated C kinase 1 partly protects mice against T. gondii infection.

Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-γ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-γ, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis.

[Gene-cloning, expression and immunoreactivity detection of Toxoplasma gondii uridine phosphorylase].

OBJECTIVE: To predict the physicochemical properties and antigenic epitopes of Toxoplasma gondii uridine phosphorylase (TgUPase), clone, and express TgUPase gene, and analyze its immunoreactivity. METHODS: The physical and chemical characters and specific epitopes of TgUPase protein were predicted by bioinformatics software tools. Total RNA was extracted from RH strain T. gondii tachyzoites. A pair of specific primers was designed according to the open reading frame of TgUPase gene (GenBank Accession No. DQ385446.1). RT-PCR product was digested with restriction enzyme and ligated into a pET-30a(+) vector. The recombinant plasmid pET-30a(+)-TgUPase was transformed into E. coli DH5alpha and the positive clones were selected by colony PCR and confirmed by double restriction enzyme digestion and sequencing. The constructed pET-30a(+)-TgUPase was then transformed into E. coli BL21(DE3) and induced with IPTG for expression. The expression product was analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with His primary antibody and human anti-T. gondii serum was used to confirm the expression of rTgU-Pase and detect its immunoreactivity. RESULTS: Bioinformatics prediction results showed that rTgUPase protein was 303 amino acids in length with a predicted molecular mass of M, 33 042.9, and this soluble protein had three potential T/B cell epitopes. The product of RT-PCR was 921 bp. Colony PCR, double restriction enzyme digestion and DNA sequencing confirmed that the recombinant plasmid pET-30a(+)-TgUPase was constructed. SDS-PAGE showed that bacteria containing recombinant plasmid pET-30a(+)-TgUPase expressed a soluble protein of His-TgUPase (about Mr 38,000) after being induced with IPTG. The recombinant protein reacted positively with His primary antibody and human anti-T. gondii serum by Western blotting analysis. CONCLUSION: The recombinant plasmid pET-30a (+)-TgUPase is constructed and the soluble rTgUPase shows immunoreactivity.

[Construction, expression and kinase function analysis of an eukaryocyte vector of rhoptry protein 17 in Toxoplasma gondii].

OBJECTIVE: To construct and express the eukaryocytic expression vector of rhoptry protein 17 of Toxoplasma gondii RH strain (TgROP17) and analyze its kinase function. METHODS: The open reading frame of TgROP17 gene was amplified from total RNA in T. gondii RH strain by RT-PCR, and cloned into p3 x Flag-CMV-14 vector to construct recombinant plasmid p3xFlag-CMV-14/TgROP17. After colony-PCR confirming, double restriction enzyme digestion and DNA sequencing, the eukaryotic expression vector p3xFlag-CMV-14/TgROP17 was transfected into HEK 293T cells. The target gene was examined by RT-PCR and the recombinant protein was detected by Western blotting. The kinase activity of TgROP17 was identified by Western blotting and its apoptotic function was assessed by flow cytometry. RESULTS: The size of RT-PCR product was 1 850 bp. The recombinant plasmid p3xFlag-CMV-14/ TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and DNA sequencing. RT-PCR and Western blotting analysis showed that TgROP17 was expressed in the p3xFlag-CMV-14/TgROPl7 transfected-HEK 293T cells rather than in mock cells. The amplified gene was with 1,850 bp and the target protein was about M, 70,000. Western blotting analysis showed that c-Jun was phosphorylated by TgROP17. Flow cytometry analysis indicated that camptothecin-induced apoptosis was inhibited by TgROP17 with an inhibition rate of 20.6% and 24.1% at 6 h and 12 h after coculture, respectively, which was higher than that of the control (P < 0.05). CONCLUSION: The eukaryotic expression vector p3xFlag-CMV-14/TgROP17 is constructed. TgROP17 has kinase activity and playes an anti-apoptosis role.

[Research progress on uridine phosphorylase in vertebrate and parasites].

Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. UPP plays an important role in the regulation of uridine homeostasis. Although UPP from a variety of organisms have many similarities in their functions, there are differences in many other aspects, such as physical and chemical properties, structure characteristics, active sites, and substrate binding sites. Therefore, UPP has broad application prospects in the design and development of antibacterial, antiparasitic drugs. This article summarizes the physico-chemical property and research progress of UPP from a variety of organisms, in order to integrate information of UPP, provide theoretic basis for further study of Toxoplasma gondii UPP protein as a feasible target antigen for toxoplasmosis vaccination.

Toxoplasma gondii protein disulfide isomerase (TgPDI) is a novel vaccine candidate against toxoplasmosis.

Toxoplasma gondii is a ubiquitous protozoan parasite that can infect all warm-blooded animals, including both mammals and birds. Protein disulfide isomerase (PDI) localises to the surface of T. gondii tachyzoites and modulates the interactions between parasite and host cells. In this study, the protective efficacy of recombinant T. gondii PDI (rTgPDI) as a vaccine candidate against T. gondii infection in BALB/c mice was evaluated. rTgPDI was expressed and purified from Escherichia coli. Five groups of animals (10 animals/group) were immunised with 10, 20, 30, 40 µg of rTgPDI per mouse or with PBS as a control group. All immunisations were performed via the nasal route at 1, 14 and 21 days. Two weeks after the last immunisation, the immune responses were evaluated by lymphoproliferative assays and by cytokine and antibody measurements. The immunised mice were challenged with tachyzoites of the virulent T. gondii RH strain on the 14th day after the last immunisation. Following the challenge, the tachyzoite loads in tissues were assessed, and animal survival time was recorded. Our results showed that the group immunised with 30 µg rTgPDI showed significantly higher levels of specific antibodies against the recombinant protein, a strong lymphoproliferative response and significantly higher levels of IgG2a, IFN-gamma (IFN-γ), IL-2 and IL-4 production compared with other doses and control groups. While no changes in IL-10 levels were detected. After being challenged with T. gondii tachyzoites, the numbers of tachyzoites in brain and liver tissues from the rTgPDI group were significantly reduced compared with those of the control group, and the survival time of the mice in the rTgPDI group was longer than that of mice in the control group. Our results showed that immunisation with rTgPDI elicited a protective immune reaction and suggested that rTgPDI might represent a promising vaccine candidate for combating toxoplasmosis.

Intranasal immunisation with recombinant Toxoplasma gondii actin partly protects mice against toxoplasmosis.

Toxoplasma gondii is a ubiquitous protozoan intracellular parasite, the causative agent of toxoplasmosis, and a worldwide zoonosis for which an effective vaccine is needed. Actin is a highly conserved microfilament protein that plays an important role in the invasion of host cells by T. gondii. This study investigated the immune responses elicited by BALB/c mice after nasal immunisation with a recombinant T. gondii actin (rTgACT) and the subsequent protection against chronic and lethal T. gondii infections. We evaluated the systemic response by proliferation, cytokine and antibody measurements, and we assessed the mucosal response by examining the levels of TgACT-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes. Parasite load was assessed in the liver and brain, and the survival of mice challenged with a virulent strain was determined. The results showed that the mice immunised with rTgACT developed high levels of specific anti-rTgACT IgG titres and a mixed IgG1/IgG2a response with a predominance of IgG2a. The systemic immune response was associated with increased production of Th1 (IFN-γ and IL-2), Th2 (IL-4) and Treg (IL-10) cytokines, indicating that not only Th1-type response was induced, but also Th2- and Treg-types responses were induced, and the splenocyte stimulation index (SI) was increased in the mice immunised with rTgACT. Nasal immunisation with rTgACT led to strong mucosal immune responses, as seen by the increased secretion of SIgA in nasal, vaginal and intestinal washes. The vaccinated mice displayed significant protection against lethal infection with the virulent RH strain (survival increased by 50%), while the mice chronically infected with RH exhibited lower liver and brain parasite loads (60.05% and 49.75%, respectively) than the controls. Our data demonstrate, for the first time, that actin triggers a strong systemic and mucosal response against T. gondii. Therefore, actin may be a promising vaccine candidate against toxoplasmosis.

[Cloning, expression and immunogenicity analysis of malate dehydrogenase gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the malate dehydrogenase (MDH) gene of Toxoplasma gondii, and analyze the immunogenicity. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii (GenBank accession No. AY650028). The coding region of TgMDH was amplified with a pair of specific primers. The product of RT-PCR was digested with double restriction enzyme and ligated into pET30a (+) vector. The recombinant pET30a (+)-TgMDH plasmid was transformed into E. coli DH5alpha. The positive clones were confirmed by the double restriction enzyme digestion, PCR and sequencing. The correct plasmid was transformed into E. coli BL21 and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Conditions for expression were optimized. Abundant soluble rTgMDH protein was purified with Ni-NTA affinity chromatography. Mice was intranasally immunized with purified rTgMDH and murine anti-rTgMDH serum was prepared. Western blotting with murine anti-rTgMDH serum and rabbit anti-T. gondii serum was used to analyze its immunogenicity. RESULTS: The product of RT-PCR was with 951 bp. The recombinant plasmid pET30a(+)-TgMDH was confirmed by the double restriction enzyme digestion, PCR and sequencing. A soluble recombinant protein with relative molecular weight of 36 000 was analyzed by SDS-PAGE, followed by coomassie blue staining. Western blotting revealed that rTgMDH can be recognized by murine anti-rTgMDH serum and rabbit anti-T. gondii serum. CONCLUSION: TgMDH gene has been expressed in prokaryotic expression system and shows immunogenicity.

[Cloning and expression of actin gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the actin gene of Toxoplasma gondii, and analyze the immunoreactivity of the recombinant protein. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgACT gene was amplified with a pair of specific primers which were designed according to the coding sequence of TgACT gene (Accession No. XM_002369622.1). The RT-PCR product was cloned into the prokaryotic expression pET-30a (+) vector. The recombinant pET30a-TgACT plasmid was transformed into E. coli DH5alpha. The positive clones were selected through the colony-PCR and confirmed by the double restrict enzyme digestion and sequencing. The correct pET30a-TgACT plasmid was transformed into E. coli BL21(DE3) and induced by IPTG. The expressed proteins were analyzed by SDS-PAGE. Western blotting assay was performed with anti-poly-histidine tag (anti-His) antibody or rabbit anti-T. gondii serum. RESULTS: The product of RT-PCR was with 1 100 bp. The recombinant plasmid pET30a-TgACT was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. SDS-PAGE results showed that the target protein was expressed in E. coli BL21(DE3) in the form of inclusion bodies with a rough molecular weight of 49 000. The purified soluble protein was obtained by using denaturation, renaturation and purification. Western blotting revealed that rTgACT can be recognized by anti-His antibody and rabbit anti-T. gondii serum. CONCLUSION: The recombinant plasmid pET30a-TgACT has been successfully constructed, and the recombinant protein TgACT is produced in E. coli and maintains specific immunoreactivity.

[Efficacy of albendazole orally administered at different dosages against Trichinella spiralis encapsulated larvae in mice].

OBJECTIVE: To evaluate the efficacy of albendazole (ABZ) orally administered at different dosages against Trichinella spiralis encapsulated larvae in striated muscle in mice. METHODS: A total of 72 BALB/c mice were divided equally into 9 groups. Each mouse was infected orally with 50 T. spiralis encapsulated larvae. At the 29th day after infection, albendazole was each orally administered to the mice of the 8 groups with doses of 50, 100, 150, 200, 250, 300, 350, and 400 mg/(kg x d), respectively, once a day at fixed time for 6 d. The control group was untreated. Mice were sacrificed at the 7th day post administration. The encapsulated larvae in diaphragmatic muscle, jugomaxillary muscle and gastrocnemius muscle were examined with pellet method. The encapsulated larva that the capsule was complete and the larva inside curled naturally with clear structure was considered survived. The therapeutic effect was estimated on the average quantity of total, survival and dead encapsulated larvae per gram muscle, total worm reduction and survival worm reduction. RESULTS: The total worm burden and survival worms showed a decreasing trend and the numbers of dead worms increased in diaphragmatic muscle, jugomaxillary muscle and gastrocnemius muscle when the dosage of albendazole were 50-250 mg/(kg x d), but the number of larvae in the muscles remained similar when the dosage of albendazole was greater than 250mg/kg x d. Compared with the control group, the total and survival worms in the muscles in 200 mg/(kg x d) and the greater dose groups decreased significantly (P<0.01). In 250 mg/(kg x d) group the total worm reduction in jugomaxillary muscle, diaphragmatic muscle and gastrocnemius muscle were 50.00%, 62.62% and 57.48%, and the survival worm reduction were 79.96%, 83.25% and 80.56%, respectively. CONCLUSION: Orally administered to mice for 6 d, albendazole at 250 mg/(kg x d) is a suitable dose against encapsulated larva stage of T. spiralis in muscle.

[Research progress in phosphoglycerate mutase].

Phosphoglycerate mutase (PGAM) is one of glycolytic enzymes, concerning with the transport of carbohydrates, metabolism, catalytic activity and growth development. PGAM was discovered in yeast firstly, and with its amino acid sequence and crystal structure determined, this protein was found in varies organism, such as human, Escherichia coli, Schistosoma japonicum and Toxoplasma gondii. This article reviews the physico-chemical property and research progress of PGAM of vertebrate, invertebrate and protozoa.

Gene-cloning, expression and antigenicity analysis of rhoptry protein 17 of Toxoplasma gondii.

OBJECTIVE: To clone and express the rhoptry protein 17 (ROP17) gene of RH strain of Toxoplasma gondii, analyze the antigenicity of recombinant protein. METHODS: Total RNA was extracted from tachyzoites of RH strain of T. gondii. The open reading frame of TgROP17 gene was amplified with a pair of specific primers which was designed according to the coding sequence of TgROP17 gene (GenBank accession No. AM075203.1), the product of RT-PCR was digested with double restriction enzyme and ligated into a pGEX-6P-1 vector. The recombinant pGEX-6P-1-TgROP17 plasmid was transferred into E. coli DH5alpha and the positive clones were selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The constructed pGEX-6P-1-TgROP17 was transformed into E. coli Rosetta (DE3) and induced with IPTG for expression. The expression products were analyzed through SDS-PAGE followed by Coomassie blue staining. Western blotting assay with GST primary antibody and rabbit anti-T. gondii serum was used to confirm the expression of GST-ROP17 and analyze its antigenic properties. RESULTS: The product of RT-PCR was with 1 850 bp. The recombinant plasmid pGEX-6P-1-TgROP17 was confirmed by colony-PCR, double restriction enzyme digestion and sequencing. A soluble recombinant protein with relative molecular weight of 96,000 was analyzed by SDS-PAGE followed by Coomassie blue staining. The GST tag in GST-ROP17 and the antigenicity of ROP17 were detected efficiently by Western blotting with the GST primary antibody and with the prepared antiserum against T. gondii, respectively. CONCLUSION: The recombinant GST-ROP17 protein has been produced in E. coli and shows specific antigenicity.

[Cloning, expression and antigenicity analysis of phosphoglycerate mutase 2 gene of Toxoplasma gondii].

OBJECTIVE: To clone and express the phosphoglycerate mutase 2 (PGAM2) gene of Toxoplasma gondii, and analyze the antigenicity of the recombinant protein. METHODS: Total RNA was extracted from T. gondii tachyzoites of RH strain and reversely transcribed into cDNA. TgPGAM2 gene was amplified by PCR and cloned into pET30a(+) vector. The constructed pET30a(+)-TgPGAM2 was transformed into E. coli DH5alpha first and selected through the colony-PCR and confirmed by the double restriction enzyme digestion and sequencing. The correct plasmid was transformed into E. coli BL21 for expression induced by IPTG and the recombinant protein was further analyzed through SDS-PAGE followed by Coomassie brilliant blue staining. Western blotting assay with rabbit anti-T. gondii serum was used to analyze its antigenicity. RESULTS: The length of PCR product was about 750 bp and the recombinant plasmid pET30a(+)-TgPGAM2 was successfully constructed. The results of SDS-PAGE and Western blotting revealed that the relative molecular weight (Mr) of the soluble recombinant protein was approximately 30 000 and could be recognized by rabbit anti-T. gondii serum. CONCLUSION: The soluble TgPGAM2 protein has been expressed in the prokaryotic expression system and maintains its antigenicity.

[Effect of oral administration of tribendimidine at different dosages against Trichinella spiralis encapsulated larvae in mice].

OBJECTIVE: To observe the efficacy of oral administration of tribendimidine (TBD) at different dosages against Trichinella spiralis encapsulated larvae in murine striated muscle. METHODS: A total of 88 BALB/c mice were divided equally into 11 groups. Each mouse was infected orally with 50 T spiralis encapsulated larvae. At day 29 after infection, TBD was each orally administered to mice of the 11 groups with doses of 0 (control group), 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 mg/(kg x d), respectively. All mice were administered once a day and lasted for 6d, and untoward drug reactions for mice were observed. Mice were sacrificed at the 7th day after administration of TBD, the encapsulated larvae in diaphragmatic muscle, jugomaxillary muscle, pectoral muscle and gastrocnemius muscle were examined by pellet method, and the total, survival and dead worms were counted. The therapeutic effect was estimated on the basis of average quantity of encapsulated larvae per gram muscle. RESULTS: During the administration period, no untoward reaction were observed in mice of 50-300 mg/(kg x d) groups. Mice in 350 and 400 mg/(kg x d) groups showed body hair dishevelment, emaciation and food-intake decrease, death rates were 25% and 50%, respectively. All mice in 450 and 500 mg/(kg x d) groups died on day 4 and 5 after TBD administration, respectively. In control group, the highest total burden (per gram) was found in diaphragmatic muscle, followed by jugomaxillary muscle, gastrocnemius muscles and pectoral muscles. TBD at dose of 50 mg/(kg x d) was unable to kill encapsulated larvae. In the rest groups, with the increase of drug dose, the total worm burden and the number of survival worms showed a decreasing trend in four kinds of muscles, and were significantly lower than that of the control group (P < 0.05 or P < 0.01). In 300 mg/(kg x d) group the number of survival worms in diaphragmatic muscle, jugomaxillary muscle, pectoral muscle and gastrocnemius muscle [8.6 +/- 1.7, 2.8 +/- 0.7, 3.9 +/- 0.8, and 0, respectively] were significantly lower than that of the control group [3648.1 +/- 989.2, 1266.4 +/- 812.3, 701.9 +/- 196.4, and 711.6 +/- 34.6] (P < 0.01). All encapsulated larvae in the four kinds of muscle died in 350 and 400 mg/(kg x d) groups. With the increase of TBD dosage, the mortality of encapsulated larvae increased in the muscles, reached up to 98.6%--100% in 300 m (kg x d) group (P < 0.01), and 100% in 350 and 400 mg/(kg x d) groups (P < .01). CONCLUSION: Oral tribendimidine administered at 50 mg/(kg x d) to mice for 6 d is unable to reduce worm burden in muscle. Tribendimidine 300 mg/(kg x d) effectively kill encapsulated larvae and is a suitable dose against encapsulated larva stage. However, tribendimidine at doses of 350 mg/(kg x d) and above for 6d is toxic to mice and even causing death.