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Objective: To examine the secretion and localization of Toxoplasma gondii Rhoptry protein 16 （ROP16） during invasion of different strains of T. gondii into host cells., Methods: The Tgrop16 gene was amplified by PCR on the cDNA of T. gondii RH strain, subcloned into the plasmid pET-32a（+）, and expressed in Escherichia coli BL21（DE3） under the induction of isopropyl β-D-1-thiogalactopyranoside. New Zealand rabbit was immuned with the expressed recombinant protein TgROP16 to produce polyclonal anti-TgROP16 antibody. The specificity and sensitivity of the polyclonal antibody were examined by Western blotting and indirect ELISA, respectively. The transcriptional and protein levels of Tgrop16 in T. gondii RH strain and Pru strain were determined by real-time PCR and Western blotting, respectively. The secretion and distribution of TgROP16 in human foreskin fibroblasts (HFFs) during the invasion by T. gondii RH strain and Pru strain were examined by indirect immunofluorescence assay (IFA)., Results: Western blotting showed a specific band at M(r) of ～100 000, indicating that the specific rabbit-derived anti-TgROP16 polyclonal antibody was capable of recognizing TgROP16. Indirect ELISA revealed a titer of 1:25 600 for the antibody. The relative expression level of Tgrop16 in Pru strain［（7.786±0.206）］ was 7 times than that in RH strain［（1.000±0.110）］（P<0.05） as detected by real-time PCR, and TgROP16 protein level was higher in RH strain than in Pru strain. IFA showed that TgROP16 was localized on the apical complex of the unrecruited tachyzoite of T. gondii before invasion and was secreted out of the recruited tachyzoite after invasion., Conclusion: The anti-TgROP16 polyclonal antibody has high specificity and sensitivity. The TgROP16 protein level is higher in the RH strain than in the Pru strain. For both strains, TgROP16 is localized on the apical complex of the unrecruited tachyzoite before invasion and secreted out of the recruited tachyzoite during invasion.

Toxoplasma gondii strains are grouped into three clonal lineages (Types I, II , and III) with different virulence. T. gondii rhoptry proteins ROP18, ROP16, and ROP5 are the major virulence factors, which are secreted into the host cell by T. gondii during invasion and mediate the strain virulence. This paper reviews the research progress of T. gondii virulence mediating factors.

Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenase XI/dispase I. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

Objective: To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization., Methods: The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21 (DE3) and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 microg purified recombinant ROP2 mixing with the same volume of Freund's adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method., Results: The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1:102400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane., Conclusion: The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

In the process of invasion and development in host cells, Toxoplasma gondii causes acute and chronic infection. The parasite manipulates the host cell elaborately and integrated to keep a delicate balance between induction and elimination of the host cell immune reaction. It can then dwell and multiply successfully in the host cell, and hopefully be transmitted to a definitive host The host cell signaling is changed and regulated extensively by the parasite in the process, which plays vital roles in parasite invasion and development. This review shed light on the manipulation of host cell signaling by T. gondii infection in these aspects: (1) T. gondii secreted proteins which manipulate host cell signaling; (2) T. gondii modulates the innate and protective immune related host cell signaling; (3) T. gondii regulates anti-apoptotic reaction and cell cycle related host cell signaling; (4) T. gondii adjusts calcium relevant host cell signaling; (5) T. gondii manipulates cell structure reorganization relevant host cell signaling.

Toxoplasma gondii is a ubiquitous, obligate intracellular protozoan parasite in human and animals. Chronic infection with this parasite is likely one of the most common infection in human. Following invasion of host cells T gondii resides within membrane-bound vacuoles known as parasitophorous vacuole (PV), which can protect the parasite from the endosomal acidification and lysosomal fusion of host cells. It plays an essential role in the whole parasitic process of T. gondii. This review summarizes the mechanism of PV formation and its function in the host cells.

To prepare a large amount of pure alive Schistosoma japonicum eggs, rabbit was infected with 2000 cercariae and its liver was taken aseptically 38-45 days after infection and homogenized. The homogenate was screened through different sieves(60, 120, 200, 300, 360 meshes per inch respectively), and washed with 1.2% NaCl. The eggs and leftover were then digested with 0.25% trypsin for 2 hours, sieved over 360 meshes per inch and washed with RPMI 1640 medium. The collected eggs reached to (95.1 +/- 6.4)% of live eggs, with a high efficiency.

Objective: To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis., Methods: The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene--green fluorescence protein (GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site (pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcription level of GFP in the tebufenozide affected yeast and the control., Results: In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tebufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tebufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control., Conclusion: The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

Objective: To screen the Schistosoma japonicum female specific expressing genes., Methods: S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms., Result: Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB017097, AF519182, M32281, and S. japonicum unknown gene AY813556 respectively., Conclusion: SSH is essential to screen the differentially expressed genes of S. japonicum female worms. A number of female specific genes have been found by this method.

Objective: To analyze the difference among antigens of Angiostrongylus cantonensis in different developmental stages and identify dominant diagnostic antigen for angiostrongyliasis., Methods: Antigens of A. cantonensis in different developmental stages were analyzed by SDS-PAGE and immunoblot., Results: The protein bands of all developmental stages were similar on SDS-PAGE. The Mr 40000, 50000, 66000 and 80000 antigens reacted not only with the sera of rats infected by A. cantonensis but also with the sera of normal rats. The Mr 104000 antigen could be discerned by sera of rats infected with A. cantonensis for 2 weeks. The Mr 32000 antigen could be recognized by sera of rats 2 weeks after infection, and the reaction became stronger with the infection continued., Conclusion: The Mr 40000, 50000, 66000 and 80000 antigens might result in the unspecific reaction in the immunodiagnosis of angiostrongyliasis using the crude antigen of A. cantonensis. The Mr 104000 of larva, Mr 33000 of adult females and Mr 32000 of the worms might be used as candidate antigens in early diagnosis and epidemiological survey of angiostrongyliasis.

Objective: To express and purify the Schistosoma japonicum adenylate kinase (AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity., Methods: The recombinant plasmid was transformed into E. coli BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS-PAGE after boiling and centrifugation. The target protein was purified with the Ni-NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti-6-His antibody, sera from rabbits 6 weeks after infected with cercariae or UV-attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum., Results: The molecular weight of the target fusion protein was Mr 40 000 after being induced with IPTG. The fused protein showed a single band when reacted with anti-6-His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum., Conclusion: The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about Mr 40000. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV-attenuated cercariae.

Objective: To construct a cDNA library from Schistosoma japonicum cercariae., Methods: Total RNA was extracted from 500,000 S. japonicum cercariae and used to synthesize the double-stranded cDNA and cloned into the TriplEx2 vector using "SMART cDNA Construction Kit"., Results: The primary titer of the constructed cDNA library is 1.8 x 10(7) pfu/ml and the titer of amplified library is 2.5 x 10(10) pfu/ml. The average size of inserts is 1.075 kb. The recombinant efficiency is 94.4%. The full length cDNA of S. japonicum TPI and JF-2 genes was successfully amplified from the library., Conclusion: A cDNA library of S. japonicum cercariae has been constructed successfully.