Pharmacokinetics of praziquantel injection in healthy buffaloes / 中国血吸虫病防治杂志

Objective To investigate the pharmacokinetics and relative bioavailability of praziquantel injection in buffaloes in contrast to praziquantel tablet. Methods A single oral administration of praziquantel tablet at a dose of 20 mg/kg or intramus-cular administration of praziquantel injection at a dose of 10 mg/kg was performed in six healthy adult buffalos according to a two-period crossover design. The praziquantel concentration in plasma was determined by a high performance liquid chromatography (HPLC)method. The pharmacokinetic parameters were calculated by non-compartmental analysis. Results The main pharma-cokinetic parameters of praziquantel tablet were as follows:Tmax=(0.60±0.29)h,Cmax=(0.57±0.37)μg/ml,T1/2β=(0.70±0.42) h,AUC=(0.80±0.70)(μg/ml)·h. The main pharmacokinetic parameters of praziquantel injection were as follows:Tmax=(0.65± 0.49)h,Cmax=(3.82 ± 1.17)μg/ml,T1/2β=(1.00 ± 0.73)h,AUC=(1.61 ± 0.89)(μg/ml)·h. The relative bioavailability of pra-ziquantel injection was 402.5％in contrast to praziquantel tablet. Conclusion The praziquantel injection has pharmacokinetic characteristics of rapid absorption,high bioavailability and extensive distribution,and the clinical recommended dosage of pra-ziquantel injection is 10 mg/kg.

Cloning, expression and characterization of a gene encoding alpha2 subunit of the proteasome in Schistosoma japonicum / 生物工程学报

The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome alpha2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTCQ the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonicum.

Cloning and expressing of cyclophilin B gene from Schistosoma japonnicum and the analysis of immunoprotective effect / 生物工程学报

The present study was intend to clone and express the cDNA encoding Cyclophilin B (CyPB) of Schistosoma japonicum, its preliminary biological function and further immunoprotective effect against schistosome infection in mice. RT-PCR technique was applied to amplify a full-length cDNA encoding protein Cyclophilin B (Sj CyPB) from schistosomula cDNA. The expression profiles of Sj CyPB were determined by Real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The cDNA containing the Open Reading Frame of CyPB was then subcloned into a pGEX-6P-1 vector and transformed into competent Escherichia coli BL21 for expressing. The recombinant protein was renaturated, purified and its antigenicity were detected by Western blotting, and the immunoprotective effect induced by recombinant Sj CyPB was evaluated in Balb/C mice. The cDNA containing the ORF of Sj CyPB was cloned with the length of 672 base pairs, encoding 223 amino acids. Real-time PCR analysis revealed that the gene had the highest expression in 18-day schistosomula, suggesting that Sj CyPB was schistosomula differentially expressed gene. The recombinant protein showed a good antigenicity detected by Western blotting. Animal experiment indicated that the vaccination of recombinant CyPB protein in mice led to 31.5% worm and 41.01% liver egg burden reduction, respectively, compared with those of the control. A full-length cDNA differentially expressed in schistosomula was obtained. The recombinant Sj CyPB protein could induce partial protection against schistosome infection.

IL-18 augments protective immunity of Sj23 plasmid DNA vaccine against Schistosoma japonicum in mice / 中国兽医学报

Two recombinant plasmids pVAX/Sj23 and pVAX/mIL-18 containing Schistosoma japonicum 23 000 membrane protein (Sj23) and murine IL-18 were evaluated for their ability to induce immune responses and to protect against S. japonicum challenge in mice. All animals vaccinated with pVAX/Sj23 alone or plus pVAX/mIL-18 developed specific anti-SWAP (soluble worm antigen preparation) ELISA antibody and splenocyte proliferation response,and co-injection of pVAX/mIL-18 significantly increased the production of IFN-γ and IL-2 compared with pVAX/Sj23 alone, indicating that IL-18 enhances the Th1-dominant immune response. The challenge experiment showed that worm reduction rates in pVAX/Sj23 group compared with control group (pVAX1) was 26.5% and in the pVAX/Sj23 plus pVAX/mIL-18 group was 41.9% ,and the hepatic egg reduction rates were 42.7 and 49.6%,respectively. These results indicated that co-injection of an IL-18 plasmid with Sj23 DNA vaccine efficiently improves the protective effect against S. japonicum infection.

Cloning and expression of Sj423 gene encoding the egg-shell precursor protein of Schistosoma japonicum / 中国人兽共患病学报

The gene fragment encoding the egg-shell precursor protein of Schistosoma japonicum was amplified with RT-PCR by using PCR primer designed according to the 423 bp cDNA fragment of the Philippine strain of S.japonicum, the corresponding mDNA fragment of Chinese strain as template and then the 5' and 3' ends of this gene cDNA were amplified with 5' RACE and 3' RACE by using a series of primers designed according to the result of sequencing. Result of sequence analysis showed that this fragment, named as Sj423, contained a complete open reading frame (ORF) of gene encoding the egg-shell precursor protein of S.japonicum.(Chinese strain). As demonstrated by sequencing analysis. No intron could be detected in this gene fragment. This gene was subsequently expressed in E.coli after cloning into the expression vector pET28c(+). The molecular mass of the expressed product of this gene was 20.9 kDa as revealed by SDS-PAGE analysis, and Western blot analysis showed that the recombinant protein expressed could react well with the rabbit antiserum against the worm antigen of S.japonicum;indicating the good antigenicity of this expressed product.

Expression of gene encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase (Sj32) and diagnostic application of recombinant protein / 中国血吸虫病防治杂志

Objective To express the gene encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase (Sj32) and evaluate the potential of the recombinant protein rSj32 in diagnosis of domestic animal schistosomiasis. Methods The DNA fragment encoding mature protease of Schistosoma japonicum asparaginyl endopeptidase was cloned with PCR from pET-28(a)/Sj32, and a recombinant plasmid was previously constructed in the laboratory, which contained the ORF of the gene encoding the pro-enzyme Sj32. The amplified DNA fragment was subcloned into pET-28a( + ) and the recombinant plasmid was transformed into E. coli BL21 (DE3) to express the mature protease of Sj32. Then the recombinant antigen (rSj32) was used in ELISA assay to diagnose schistosomiasis of mice, rabbits and water buffalo artificially infected. The detection effects of soluble Schistosoma japonicum egg antigen (SEA) , rSj32 and the recombinant 23 KDa membrane protein were compared. Results The recombinant antigen rSj32 with a molecular weight 41 KDa was successfully produced in E. coli BL21 ( DE3) and was purified with His Column with a yield of 25 mg/L E. coli culture. By using rSj32 as coating antigen in ELISA assay to detect the specific antibody in artificially infected mice, rabbits and buffalo, the sensitivities were 88.9% , 85.0% and 71.8% , respectively, the specificities were 100% , 96.7% and 96. 9% , respectively. There were no significant differences among the detection results of rSj32, SEA and rSj23. Conclusion rSj32 is a promising antigen for serological diagnosis of domestic animal schistosomiasis.

Screening of T7 phage-display cDNA library from liver of Microtus fortis with extracts of schistosomulum and characterization of the positive clones / 生物工程学报

Microtus fortis is naturally resisitent to Schistosoma japonicum. In order to find schistosome-resistence-related genes of Microtus fortis, a T7 phage-display cDNA library from liver of Microtus fortis was screened with the soluble lysate of schistosomula. The specific phages were enriched 375-fold after 3 rounds of biopanning. Ninety-two positive clones picked at random were sequenced and 19 ESTs including 6 unreported genes were obtained. Compared with the negative phage clone control, five positive clones, No.4 (GenBank Accession No.: EW968294), No.13 (GenBank Accession No.: EW968303), No.14 (GenBank Accession No.: EW968304), No.15 (GenBank Accession No.: EW968305) and No.18 (GenBank Accession No.: EW968308) could induce significantly higher schistosomula mortality rate when co-cultivated with schistosomula. According to the function analysis and the shistosomula-killing effect in vitro, the genes encoding CASP8 and FADD-like apoptosis regulator isoform protein, alpha-2-HS-glycoprotein, M4 protein, R3H domain (binds single-stranded nucleic acids) isoform 2 and 3 previously unreported proteins (No.14, No.15 and No.18) obtained here, were schistosomiasis-resistence-related genes of Microtus fortis.

Cloning, expressing and characterizing of a phosphoglycerate mutase gene of Schistosoma japonncum / 生物工程学报

Phosphoglycerate mutase (PGAM) is a key enzyme in glycolytic pathways. With PCR technique based on an EST identified in our lab, a novel gene named SjPGAM (GenBank Accession No. EU374631) was cloned. Sequence analysis revealed that the ORF of SjPGAM gene contained 753 nucleotides, encoding 250 amino acids, and the molecular weight was about 28.26 kD. Real-time PCR analysis showed that the mRNA level of SjPGAM was much higher in the 14 days and 19 days schistosomula than other stages, suggesting that the gene was a schistosomula stage differential expression gene. The SjPGAM cDNA fragment was subcloned into an expression vector pET-28a (+) and transformed into Escherichia coli BL21 cells. In the presence of IPTG, the 31 kD fusion protein was expressed in included bodies. Western blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the mechanism of the PGAM in the glycolytic pathways of Schistosoma japonnicum.

Observations on protective immunity in mice induced by recombinant Schistosoma japonicum Sj14FABP through several adjuvant formulations / 中国血吸虫病防治杂志

Objective To test the protective immunity in mice induced by recombinant Schistosoma japonicum Sj14FABP through several adjuvant formulations. Methods The recombinant Schistosoma japonicum Sj14FABP was prepared by expression in E. coli as a GST fusion protein (rSj14/GST) and used to vaccinate outbred Kunming mice by using complete Freund's adjuvant (FCA)/incomplete Freund's adjuvant (FIA), Bacillus Calmette-Guerin (BCG) and the immunostimulating complex (ISCOM) as adjuvant respectively. Results The purified recombinant protein rSj14/GST was immunogenic in mice, and 34.3% and 36.0% worm reduction rates were obtained in outbred Kunming mice immunized intradermally with BCG adjuvant and immunized subcutaneously with ISCOM adjuvant respectively, compared with non-vaccinated control group. However, intramuscularly vaccination with rSj14/GST in FCA/FIA was not protective, although the high level of IgG antibody was induced. Conclusion Both BCG and ISCOM are suitable adjuvants for rSj14/GST.

Construction of Phage Display cDNA Library from Adult Worms of Schistosoma japonicum / 中国寄生虫学与寄生虫病杂志

Objective To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum.Methods Total RNA was extracted from adult worms of S.japonicum by Trizol reagent and mRNA was isolated from the total RNA.The ds cDNA was synthesized by reverse transcription using random primer.Directional EcoRⅠ/HindⅢ linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoRⅠand HindⅢ,which resulted in ds cDNA with EcoRⅠand HindⅢ adhering ends.The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector.After packaging in vitro,the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library.Plaque assay and PCR were used to evaluate the library.Seven known objective genes of S.japonicum were screened by PCR to detect the representation of the library.Result Primary library capacity was 4.98?106 pfu,and the titer of amplified library was 3.85?1011 pfu/mL.The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%,in which 95.6% inserted cDNA fragments were longer than 300 bp in length.All the seven known objective genes of S.japonicum were amplified from the library.Conclusion The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

Construction of a T7 phage display cDNA library from lung of Microtus fortis / 中国血吸虫病防治杂志

Objective To construct a T7 phage display cDNA library from the lung of Microtus fortis for further screening the schistosomiasis-resistence-related genes of Microtus fortis. Methods mRNA was isolated from total RNA extracted from the lungs of Microtus fortis by TRIzol reagent, and was used to synthesize double strain cDNA by the reverse transcription. Then the double strain cDNA was given with EcoRⅠ and Hind Ⅲ adhering ends by ligation with the directional EcoRⅠ/Hind Ⅲ linkers and digestion with EcoRⅠ and Hind Ⅲ. The double strain cDNA fragments longer than 300 bp in length were fractionated by the Mini Column, and ligated into the T7 Select 10-3b vector with EcoRⅠ and Hind Ⅲ adhering ends. After packaging in vitro, the recombinant T7 Select 10-3b was transformed into BLT5403 to construct a T7 phage display cDNA library. Results The library constructed here contained 1.5?106 clones and the titer of the amplied library was 1.1?1012 pfu/ml. The PCR identification results of 100 clones picked at random showed that 91% clones were recombinant and 90% of recombinant clones contained cDNA fragments longer than 300 bp in length. Conclusion A T7 phage display cDNA library from the lung of Microtus fortis is successfully constructed.

Screening the target genes of Schistosoma japonicum related to the naturely resistance of Microtus fortis / 中国血吸虫病防治杂志

Objective To look for the genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis.Methods The fresh sera of Microtus fortis were used to screen a T7 phage display cDNA library from worms of Schistosoma japonicum established in our lab.The positive clones were sequenced and functionally analysed through bioinformatics.Results The specific phages binding to the sera of Microtus fortis were enriched 857-fold after three rounds of biopanning,and 58 positive clones picked at random were sequenced and 10 ESTs were obtained.BLASTn results showed that 7 ESTs had 99%-100% similarity to the genes of Shistosoma japonicum reported in GenBank and 1 EST had 82% similarity to a zinc finger protein encoden gene from Pan troglodytes.The results of these ESTs function prediction indicated most of them were involved in the regulation of gene expresion of Schistosoma japonicum.Conclusions Several target genes of Schistosoma japonicum related to the Schistosoma-resistance of Microtus fortis are obtained and those would lay foundation to expatiate the native resistance mechnism of Microtus fortis to Schistosoma japonicum.