日本血吸虫急性感染小鼠血清肽谱分析

目的分析急性感染日本血吸虫小鼠血清多肽,寻找具有潜在诊断意义的标志蛋白。方法构建日本血吸虫急性感染小鼠模型,利用螯合铜离子富集纯化血清多肽,应用基质辅助激光解吸电离时间飞行质谱(MALDI-TOF-MS)Flex Analysis分析技术结合ClinProTool建模方程进行分析。结果与未感染对照组小鼠血清多肽指纹图谱比较,在0.8～17.0ku区段,日本血吸虫急性感染小鼠9.068、2.082、4.533ku多肽表达上调(P<0.01),3.493、2.869、2.024、4.965ku多肽表达下调(P<0.05)。结论日本血吸虫感染血清9.068、2.082、4.533ku多肽作为生物标志物对日本血吸虫病早期诊断有潜在应用价值。

细胞因子对感染日本血吸虫小鼠肝纤维化的影响

目的探索细胞因子对日本血吸虫病肝纤维化的影响。方法用免疫组化技术，观察１１０只昆明鼠（其中９０只为感染日本血吸虫鼠）肝内肿瘤坏死因子（ＴＮＦ）α和细胞外基质的量及分布，和注射ＴＮＦ及白细胞介素（ＩＬ）１后它们的变化。结果感染后１２周小鼠肝内ＴＮＦ达高峰，主要分布在虫卵肉芽肿内及其周围。纤维连接素、层连素、Ⅰ及Ⅲ型胶原分别于感染后１６及２０周达高峰，由细线状增宽变厚，呈条索状沉积在肉芽肿内及其周围。注射ＴＮＦ后，感染鼠肝内Ⅰ型胶原明显增加（Ｐ＝００１）；注射ＩＬ１使Ⅰ及Ⅲ型胶原略多于同期未注射组；联合注射ＴＮＦ和ＩＬ１，肝内ＴＮＦ及Ⅰ型胶原显著高于同期非注射组（Ｐ＜００５，Ｐ＝００１）。结论ＴＮＦ可刺激昆明鼠肝内胶原增加，ＩＬ１具有协同ＴＮＦ的作用。

Cloning and Functional Analysis of the Full Length cDNA Sequence of Eukaryotic Translation Initiation Factor 5 in Schistosoma japonicum

The expressed sequence tag of eukaryotic translation initiation factor 5 （eIF5） from the Schistosoma japonicum adult worm cDNA library through subtractive hybridization between male and female worms was analyzed by the bioinformatics method. The overlapping sequences were assembled into one that includes the complete open reading frame （GenBank accession number： AY686501）. The full-length cDNA of SjeIF5 was cloned into a pET-28c^（＋） vector, which generated a prokaryotic expression plasmid, and a fusion protein of 18 kDa was induced in Escherichia coll. The recombinant expression of eIF5 protein of Schistosoma japonicum was purified. The immunoprotection test against schistosomiasis demonstrated that the recombinant protein worked to a certain extent, especially in the reduction of eggs in the liver of the host.

Construction of a hepatic stellate cells subtracted cDNA library of differentially expressed genes in normal mice and mice with Schistosomiasis japonica

To construct a hepatic stellate cells (HSCs) subtracted cDNA library to find differentially expressed genes in normal mice and mice infected with Schistosoma japonicum (S. japonicum). Suppression subtractive hybridization (SSH) was used. The cDNA fragments of normal mouse were compared to those of schistosoma-infected mice to find differentially expressed genes. Then differentially expressed cDNA fragments were directly inserted into T/A cloning vector to set up the subtractive library. Amplification of the library was carried out with transformation of DH5α. The amplified library contained more than 400 positive bacterial clones, which were then hybridized with forward and backward subtracted probes for differential screening. One hundred positive bacterial clones were randomly selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. The subtracted cDNA library of differentially expressed genes of HSCs was constructed successfully, the library is efficient and lays foundation for screening and cloning new and specific genes of schistosomiasis.

Selection of the Mimic Epitopes of Antigens from Schistosoma japonicum by Using Phage Display Techniques

To obtain short peptides simulating antigenic epitopes related to natural resistance against Schistosoma japonicum (S.j) in rats, and to explore their immune protection against S.j in mice, phage random peptide library of 12 amino acids were screened with purified IgG from normal rat sera. Positive clones that were obtained after three rounds of biopanning were detected by ELISA, and two of them were sequenced. Kunming mice were immunized with mixed phage clones. Each mouse was challenged with 40±1 S.j cercariae, and all mice were perfused 45 days post-challenge. The worms and the liver eggs were counted. The results were that the specific phages binding to IgG were enriched 300 folds after three rounds of biopanning. Twenty clones were detected by ELISA and 19 of them bound to the specific IgG of rat sera. The sequence of two clones revealed no homology with other sequences in the GenBank. Compared with the control groups, the reduction rates of the worm burden and liver egg were 33.6% and 59.8%, respectively. It was concluded that the specific peptides, which simulate antigenic molecules correlated with natural resistance to S.j in rats could be obtained by immunosceening phage random peptide library and a protective immunity against S.j can be detected by these epitopes in mice.

Expression and Analysis of Microtus fortis Against Schistosoma japonicum CD72

The total RNA was extracted from Microtus fortis liver tissue which before being infected and after being infected 10 d and 15 d by the Schistosoma japonicum cercariae. Using rattus norvegicus CD72 gene probes were used to hybridize analysis of CD72 difference expression in the Microtus fortis liver tissues which were infected with Schistosoma japonicum before and after being infected. At the same time, the cDNA sequence and encoded amino acid sequence of the rattus norvegicus CD72 gene and CD72 protein structural domains were analyzed by using bioinformatics. The results showed that the CD72 expression levels in the liver tissue of Microtus fortis after being infected was significantly higher than before being infected. The rattus norvegicus CD72 cDNA sequence of a total length is 1479 bp and encode 364 amino acid residues and rattus norvegicus CD72 protein containing a CD72 superfamily domain.

Expression and Analysis of Microtus fortis against Schistosoma japonicum CD36 Gene

The total RNA was extracted from Microtusfortis liver tissue which before being infected and after being infected 10 d and 15 d by the Schistosoma japonicum cercariae. Using Rattus norvegicus CD36 gene probe to hybridize analysis of CD36 difference expression in the Microtus fortis liver tissues which were infected with Schistosorna japonicum before and after being infected. At the same time, the cDNA sequence and encoded amino acid sequence of the Rattus norvegicus CD36 gene and CD36 protein structural domains were analysized by using bioinformatics. The results showed that the CD36 expression levels in the liver tissue of Microtus fortis after being infected were significantly higher than before being infectied. The Rattus norvegicus CD36 cDNA sequence of a total length is 1625 bp and encoded 472 amino acid residues and Rattus norvegicus CD36 protein containing a CD36 superfamily domain.

Screening and Primary Characterization of NewAntigen Genes of Schistosoma Japonicum

To find Schistosoma japonicum(S.j)new antigen gene thus provide more useful vaccine candidates, the cDNA library of S.j adult worm was screened with sera of rabbits immunized with the membrane antigens of Schistosoma japonicum hepato-portal schistosomula (SjHmAg). The positive clones were amplified by PCR and sequenced, then the sequences of clones were compared with all sequences in GenBank database using Blast process. The new clones were submitted to GenBank for accession numbers. Fifteen positive clones were obtained after three rounds of immunoscreening. The size of S.j cDNA fragments in positive clones ranged from 0.7?kb-3.0?kb after automatically excised with the helper phage. Sequence analysis revealed that partial sequence of clone M5 had significant homology with S.j mitochondria mRNA, the other positive clones were new S.j genes. M2 clone sequence (GenBank accession number AF502579) was 730?bp long it had a 117?bp open reading frame (ORF). The sequence of M15 (GenBank accession number AF502582) has no transmembrane region and encodes 92 amino acids, and its protein contains a ferredoxins iron-sulfur binding region signature and two VWFC signal regions. The size of M1、M8、M9、M12(GenBank accession numbers: AF502578, AF502580, AF500622, AF502581) ranges from 402?bp to 766?bp. It concluded that the sera from rabbit immunized with SjHmAg could recognize S.j specific antigens molecules, and these antigens may induce the protective immunity against S.j infection.

Selection of Immunogenic Peptide Mimics of Male Worm Origin of Schistosoma Japonicum using Phage Display Peptide Library

To select the immunogenic short peptide mimics of male worm origin of Schistosoma japonicum (Sj) and to explore their protection effect against schistosomiasis in mice, the random phage display peptide library of 12-mer was screened with IgG to soluble male worm antigen of Sj, and the specific positive clones selected through three rounds of screenings were detected by Dot-ELISA, and then injected subcutaneously into mice for vaccination and protection assessment against Sj. It was found that 18 randomly picked phage displayed clones all showed definite antigenicity with various intensities. The pooled phages displayed clones could induce production of specific antibodies and cause 31.72% of worm reduction rate and 51.54% of egg reduction rate in mice, revealing a significant difference (P<0.001) in comparison with those of the controls. It concludes that the short peptide mimics of male worm origin of Sj obtained by affinity screening phage display peptide library can elicit partial protection against this pathogen.

Dynamic changes in the collagen metabolism of liver fibrosis at the transcription level in rabbits with Schistosomiasis japonica

OBJECTIVE: To study the role of the synthesis and degradation of collagen at the transcription level during liver fibrogenesis due to schistosomiasis japonica in rabbits. METHODS: New Zealand rabbits challenged by cercariae of Schistosoma japonicum (S. japonicum) were served as animal models for liver fibrosis. Liver specimens were collected through operations at 4, 6, 8, 10, 12, 16, 20, 24 and 28 wks after challenge. Type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels of liver tissue were detected by RT-PCR + Dot blot. The size of egg granulomas and the degree of liver fibrosis were measured by histopathological examinations. RESULTS: Type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased simultaneously in the early stage after challenge. Most of them reached their peak at 10 weeks, and compared with normal controls, type I collagen, type III collagen, type IV collagen, MMP-1 and MMP-9 mRNA levels increased by 12.0-, 11.0-, 6.6-, 10.0- and 11.0-fold, respectively, coinciding with the change of egg granulomas, i.e., the change in the inflammatory process. Then both collagen and collagenase mRNA levels decreased. Type I, III and IV collagen mRNA levels declined to 2-fold to 3-fold as compared with normal controls (P 0.05) at 28 wks. This study shows that the synthesis and degradation of collagen keep a dynamic balance at the early stage of schistosomiasis japonica challenge, while at the later stages the quantity of collagen synthesis was higher than that of collagen degradation. CONCLUSIONS: It was confirmed at transcription level that when the quantity of collagen synthesis was higher than that of collagen degradation liver fibrogenesis may be resulted in.

Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli

OBJECTIVE: To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli. METHODS: SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase-polymerase chain reaction (RT-PCR) with total RNA extracted from adult worms of S. japonicum. The RT-PCR product was cloned into T vector and sequenced. The SjcTM cDNA, derived from the constructed TA clone pGEM-SjcTM, was then subcloned into the expressing vector pBV220. After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature-dependent condition. RESULTS: The RT-PCR product, cloned into a T vector, was sequenced and shown to be 96.5% identical at the nuclei acid level and 98.1% identical in deduced amino acid sequence to that of S. mansoni tropomyosin. The target DNA fragment was then subcloned into a prokaryotic vector pBV220. Induced expression in E. coli DH5alpha cells resulted in a constant level of recombinant protein production. The results of SDS-PAGE and Western blot revealed that the molecular weight of non-fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S. japonicum tropomyosin (SjcTM). CONCLUSION: The engineering of the cDNA encoding S. japonicum tropomyosin and its bacterial expression was successfully made.

Generation and analysis of 113 adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags

OBJECTIVE: To rapidly and economically obtain knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes using expressed sequence tag (EST). METHODS: A directional cDNA library constructed from Schistosoma japonicum (Chinese strain) adult stage RNA was used to generate expressed sequence tags (ESTs). These were compared against an EMBL-parasites database and GENBANK database by BLASTn and BLASTx. RESULTS: A total of 314 phage clones were randomly selected for generating expressed sequence tags (ESTs). From these clones, 132 EST-quality sequence were obtained. Among these EST-quality sequences, 113 ESTs were successfully submitted to the dbEST at GenBanK. A total of 7.6% of these EST-quality sequences were previously identified sequence of Schistosoma japonicum, while 4.5% were putatively identified sequences of Schistosoma japonicum. A total of 23.5% of these EST-quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms. 57.6% had no matches in the database and were classified as unknown sequences. Most ESTs with the putative protein identified belonged to housekeeping proteins. Information about several interesting genes was found. CONCLUSION: Partial cDNA sequencing to generate expressed sequence tags (ESTs) has the potential to rapidly and economically increase our knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes.

Cloning of cDNA encoding Schistosoma japonicum tropomyosin and its expression in Escherichia coli

To perform cloning of the gene encoding Chinese Schistosoma japonicum tropomyosin (SjcTM) and its expression in Escherichia coli Methods SjcTM cDNA fragment, except for 14 amino acids at the amino terminus, was obtained by reverse transcriptase polymerase chain reaction (RT PCR) with total RNA extracted from adult worms of S japonicum The RT PCR product was cloned into T vector and sequenced The SjcTM cDNA, derived from the constructed TA clone pGEM SjcTM, was then subcloned into the expressing vector pBV220 After characterization by agarose gel electrophoresis, endonucleases digestion and PCR, the resultant recombinant plasmid was used for expression under the temperature dependent condition Results The RT PCR product, cloned into a T vector, was sequenced and shown to be 96 5% identical at the nuclei acid level and 98 1% identical in deduced amino acid sequence to that of S mansoni tropomyosin The target DNA fragment was then subcloned into a prokaryotic vector pBV220 Induced expression in E coli DH5α cells resulted in a constant level of recombinant protein production The results of SDS PAGE and Western blot revealed that the molecular weight of non fusion recombinant protein (rSjcTM) was approximately 32 kDa and could be recognized specifically by a polyclonal antiserum specific for native S japonicum tropomyosin (SjcTM) Conclusion The engineering of the cDNA encoding S japonicum tropomyosin and its bacterial expression was successfully made

Generation and analysis of 113 adult stage Schistosoma japonicum (Chinese strain) expressed sequence tags

To rapidly and economically obtain knowledge about adult stage Schistosoma japonicum (Chinese strain) expressed genes using expressed sequence tag (EST) Methods A directional cDNA library constructed from Schistosoma japonicum (Chinese strain ) adult stage RNA was used to generate expressed sequence tags(ESTs) These were compared against an EMBL parasites database and GENBANK database by BLASTn and BLASTx Results A total of 314 phage clones were randomly selected for generating expressed sequence tags(ESTs) From these clones, 132 EST quality sequence were obtained Among these EST quality sequences,113 ESTs were successfully submitted to the dbEST at GenBanK A total of 7 6% of these EST quality sequences were previously identified sequence of Schistosoma japonicum, while 4 5% were putatively identified sequences of Schistosoma japonicum A total of 23 5% of these EST quality sequences were putatively identified sequence of Schistosoma mansoni or other organisms 57 6% had no matches in the database and were classified as unknown sequences Most ESTs with the putative protein identified belonged to housekeeping proteins Information about several interesting genes was found Conclusion Partial cDNA sequencing to generate expressed sequence tags (ESTs) has the potential to rapidly and economically increase our knowledge about adult stage Schistosoma japonicum(Chinese strain) expressed genes

A Mathematical Model with Delays for Schistosomiasis Japonicum Transmission

A dynamic model of schistosoma japonicum transmission is presented that incorporates effects of the prepatent periods of the different stages of schistosoma into Baxbour＇s model. The model consists of four delay differential equations. Stability of the disease free equilibrium and the existence of an endemic equilibrium for this model are stated in terms of a key threshold parameter. The study of dynamics for the model shows that the endemic equilibrium is globally stable in an open region if it exists and there is no delays, and for some nonzero delays the endemic equilibrium undergoes Hopf bifurcation and a periodic orbit emerges. Some numerical results are provided to support the theoretic results in this paper. These results suggest that prepatent periods in infection affect the prevalence of schistosomiasis, and it is an effective strategy on schistosomiasis control to lengthen in prepatent period on infected definitive hosts by drug treatment （or lengthen in prepatent period on infected intermediate snails by lower water temperature）.

A novel and effective hyperthermia method for Schistosomiasis japonica prevention and treatment

The emerging resistance to schistosoma has been paid much attention and is in urgent need for a novel strategy to control the prevalent parasitic zoonosis. In this study, the efficiency of hyperthermia therapy was investigated by the skin hyperthermia device. The survival rate of cercariae decreased from 68.15 % （37 ℃, 5 min） to 0 （49℃, 10 min） with the thermal dosages increased, which proved the preventing effect of hyperthermia therapy （P 〈 0.05）. Therapeutic effects were assessed in Schistosoma japonicum-infected BALB/c mice. When the cercarial contact region of the skin was treated at 45-49℃ for 5 min within 8 h of infection, worm reduction rate （WRR） reached 74 %-83 % （P 〈 0.01）. The sensitivity of adult schistosoma to heat was also investigated using microwave intraperitoneal hyperthermia （thermal dosages 42-43℃, 20 min）. The WRR, hepatic shift rates and egg reduction rates were 23.7 %, 40 % and 30 %, respectively, comparing with 80.2 %, 59.6 % and 53.9 % of praziquantel （PZQ）-treated group. Encouraging results have been obtained that hyperthermia can effectively kill schistosomula, especially with the appearance of cercarial dermatitis, while PZQ lacks efficacy against the cercariae. Thus, hyperthermia therapy would show significant benefit in preventing and treatment of schistosoma, especially in the early stage.