[Cloning and characterization of conservative region of tyrosine kinase 4, a novel gender-associated gene of Schistosoma japonicum].

OBJECTIVE: To clone and express the conservative region of gene encoding tyrosine kinase 4 of Schistosoma japonicum and identify the difference in gene expression between genders of S. japonicum. METHODS: The gene fragment was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the total RNA isolated from adult S. japonicum (Chinese strain) with primers designed according to SmTK4 encoding tyrosine kinase 4. The purified PCR product was ligated with pET28a and the recombinant protein was induced to express, and analyzed by SDS-PAGE, Western blotting and tools of bio-informatics. Subsquently, total RNA was respectively isolated from adult males, females and both worms of S. japonicum. The real-time PCR was performed with corresponding primers after reverse transcription to show the expression levels of the gene in both genders. RESULTS: A 582 bp in size of the DNA fragment was acquired by RT-PCR. Sequence analysis indicated that the fragment showed 91% in homology to that of SmTK4, and the deduced amino acid sequence showed to be 98% identical with that encoded by SmTK4. SDS-PAGE analysis revealed that the relative molecular weight (M(r)) of expressed protein rSjTK4 was approximately 26000. The bio-information analysis demonstrated that the protein had multiple sites of enzymatic activities. The relative number of copies of SjTK4 in male worms was 0.61 +/- 0.29, while 0.03 +/- 0.02 in female worms, showing that the mRNA level of TK4 in male worms was 18 times higher than that in females. CONCLUSION: The conservative region of gene encoding tyrosine kinase 4 of S. japonicum is successfully cloned and expressed. The mRNA level of TK4 in male worms is significantly higher than that in females.

[Cloning, expression and immunodiagnostic evaluation of antigen EPC1 from Echinococcus granulosus].

OBJECTIVE: To clone and express EPCl gene of Echinococcus granulosus, and investigate its immunogenicity and diagnostic value. METHODS: Total RNA was extracted from hydatid cyst protoscoleces and EPC1 gene of Echinococcus granulosus was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector, and then subcloned into the prokaryotic expression vector PET28a(+). The positive recombinants were transformed into Escherichia coli BL21 (DE3), and followed by expression of the protein induced by IPTG. The recombinant protein was identified by SDS-PAGE and Western blotting, and used to establish ELISA. Serum samples from patients with cystic echinococcosis (60 cases), alve-olar echinococcosis (37 cases), cysticercosis (16 cases), clonorchiasis sinensis (7 cases), schistosomiasis japonica (4 cases) and healthy persons (33 cases) were examined. RESULTS: The recombinant plasmid PET28a-EgEPC1 was identified by restriction enzyme digestion and sequencing. SDS-PAGE result showed that the recombinant containing recombinant plasmid PET28a-EgEPC1 expressed a soluble fission protein of EgEPC1 (about M, 11 000). The protein was recognized by pool sera of cystic echinococcosis patients. The overall sensitivity and specificity of diagnosis by ELISA for cystic echinococcosis were 78.3% (47/60), and 98.3% (59/60), respectively. The cross reaction with sera of alveolar echinococcosis was 40.5% (15/37). CONCLUSION: The recombinant EgEPC1 antigen has diagnostic value in cystic echinococcosis.

[Preliminary study on the immunological characteristics of permissive and non-permissive hosts infected with Schistosoma japonicum].

OBJECTIVE: To study the difference among immune responses of three kinds of experimental animals with different susceptibility to the infection of Schistosoma japonicum, and preliminarily explore the mechanism of the immune response in permissive and non-permissive hosts. METHODS: Twelve animals of each kind of rodents, C57BL/6 mice, Sprague Dawley (SD) rats and Microtus fortis, were randomly divided into the infected group and uninfected group each with 6 animals. In infected groups of C57BL/6 mice, SD rats, and M. fortis, each animal was infected with 20, 200 and 1000 cercariae of S. japonicum, respectively. 42 d later, all rodents were sacrificed. Adult worms in portal vein and granulomas in liver were observed and the sera were collected. The levels of cytokines IL-10 and IFN-gamma as well as serum IgG, IgG2a, and IgG1 were detected by ELISA. RESULTS: At the 42th day post infection, worms in portal vein and liver granulomas were observed in C57BL/6 mice and SD rats, but not in M. fortis. The level of IL-10 in the sera of SD rats [(2.21 +/- 0.12) pg/ml] was significantly higher than that in the sera of M. fortis [(1.64 +/- 0.39) pg/ml] and C57BL/6 mice [(0.10 +/- 0.04) pg/ml] (P<0.01). IL-10 in the sera of M. fortis was also significantly higher than that in the sera of C57BL/6 mice (P<0.01). IFN-gamma in the sera of SD rats [(0.21 +/- 0.11) pg/ml] was significantly higher than that in the sera of M. fortis [(0.11 +/- 0.03) pg/ml] and C57BL/6 mice [(0.09 +/- 0.02) pg/ml] (P<0.05), but no difference between M. fortis and C57BL/6 mice (P>0.05). The levels of IgG (1.53 +/- 0.31), IgG1 (1.48 +/- 0.44) and IgG2a (0.41 +/- 0.11) in SD rats were significantly higher than that in the sera of M. fortis (0.48 +/- 0.14, 0.15 +/- 0.03 and 0.12 +/- 0.061) (P<0.01). The levels of IgG (1.21 +/- 0.16), IgG1 (0.88 +/- 0.31) in C57BL/6 mice were significantly higher than that in the sera of M. fortis (P<0.01). IgG1 antibody is the predominant subclass in the three kinds of rodents. The levels of IL-10, IFN-gamma and antibody subclass IgG, IgG1, IgG2a in all non-infected rodents were not detected. CONCLUSION: IL-10 in non-permissive hosts, which is an essential agent in the regulation of Th2 immune response, is higher than that in permissive host It may play an important role in the resistance to schistosome in the non-permissive hosts.

[Cloning, expression and analysis of the heat shock protein of Cryptosporidium andersoni].

OBJECTIVE: To clone and express the partial encoding sequence of Mr 70,000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein. METHODS: Total RNA was extracted from oocysts of C. andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively. RESULTS: The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70,000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43,000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization. CONCLUSION: The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.

[Isolation and identification of an isolate of cow-origin Cryptosporidium sp].

OBJECTIVE: To isolate and identify Cryptosporidium oocysts from feces of naturally infected cow. METHODS: Fecal samples were collected from Cryptosporidium infected cows confirmed by modified acid-fast staining method. Oocysts were isolated and purified with Sheathed sucrose density gradient centrifugation technique. Genomic DNA was isolated with Chelex-100. Both primers were designed to amplify Cryptosporidium small subunit ribosome RNA gene (SSU rRNA) and Cryptosporidium oocyst wall protein gene (COWP), respectively. The PCR products were cloned into pGEM-T and pGEM-T Easy vector and sequenced subsequently. Homology and phylogeny were analyzed with BLASTn and MEGA software. RESULTS: The results suggested that the size of oocysts was (7.4+/-0.32) microm by (5.4+/-0.21) microm and the ratio of length and width was 1.37+/-0.07 (n=20). BLASTn revealed that the identity of SSU rRNA and COWP gene of Cryptosporidium isolated from cow to the counterparts of andersoni was 100% and 99% respectively. Phylogenetic reconstruction placed the isolated Cryptosporidium within the C. andersoni clade based on the sequence of SSU rRNA and COWP gene. CONCLUSION: What isolated from naturally infected cow feces has been identified as C. andersoni.

[Protective immunity induced by recombinant Schistosoma japonicum thioredoxin in mice].

OBJECTIVE: To investigate the protective immunity of the recombinant thioredoxin of Schistosoma japonicum(reSjcTrx)in mice. METHODS: Thirty 6-week old female C57BL/6 mice were randomly divided into 3 groups with 10 each: reSjcTrx with Montanide ISA720 adjuvant, adjuvant control, and infection control. Mice were vaccinated subcutaneously at week 0, 2, 4 with reSjcTrx emulsified in Montanide ISA720 adjuvant. The mice in adjuvant group was injected three times with Montanide ISA720 and saline only. Mice in infection control group were given no injection. Three weeks after final injection, each mouse was challenged with 30 +/- 1 cercariae of S. japonicum (Chinese strain). At the week six after challenge, all mice were sacrificed and perfused. The number of recovered worms and eggs from liver tissue of mice were counted. Sera were collected from mice before immunization, before challenge and before killing. The anti-SjcTrx antibodies in sera were detected with ELISA. RESULTS: ELISA showed a high level of specific IgG antibodies in mice immunized with the reSjcTrx. The worm reduction rate and egg reduction rate of reSjcTrx immunization group were 22.8% and 29.5% respectively, significantly higher than those of the control groups (P < 0.05). SDS-PAGE and Western blotting revealed that the molecular weight of expressed protein was around Mr 14 000 and could be recognized by sera from rabbit infected with S. japonicum and from mice immunized with reSjcTrx. CONCLUSION: The reSjcTrx induces certain protective immunity against schistosomiasis japonica in mice.

[Preparation of DNA from Cryptosporidium parvum oocysts for PCR detection].

OBJECTIVE: To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR. METHODS: After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C. parrum gene (L.16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex 100 method was also tested by PCR. RESULTS: One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst. CONCLUSION: The three kinds of extraction can all be served as templates for PCR detection of C. parvum oocysts, while Chelex 100 method is simpler, quicker and more reliable for DNA extraction of the parasite.