Molecular cloning and characterization of a novel serpin gene of Clonorchis sinensis, highly expressed in the stage of metacercaria

Yang, Yu-Chih; Hu, Dong; Wang, Lexun; Liang, Chi; Hu, Xuchu; Wang, Xiaoyun; Chen, Jingfang; Xu, Jin; Yu, Xinbing

doi:10.1007/s00436-009-1654-z

Cited by 18 publications

(13 citation statements)

References 17 publications

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“…The construction of C. sinensis metacercaria cDNA library was described previously (Yang et al 2009). About 3,475 unigenes were obtained, in which a complete sequence coding C. sinensis metacercaria glycerol 3-phosphate dehydrogenase (GPD) was recognized by BLASTx in NCBI (http://www.ncbi.nlm.nih.gov).…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant protein was eluted with 100 mM imidazole, and then imidazole was removed from protein by dialysis using phosphate-buffered to manufacturer's instructions and treated with DNase (Promega, USA), PCR was employed to amplify the transcripts of GPD (606 bp) and C. sinensis β-actin (GenBank accession No. EU109284, 323 bp), which was used as a positive control (Yang et al 2009;Yoo et al 2009). The sense and antisense primers for CsGPD were 5′-TATCGGGTCGGGAAACTGGG-3′, 5′-ACGGCA ACAATGTTCTTCAAAGC-3′, and for β-actin of C. sinensis were 5′-GGTGACGCTGAAGTATCCTATTG A-3′, 5′-CCAAAGCATAGCCCTCGTAGAT-3′, respectively.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Molecular cloning, expression, and immunolocalization of the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis

et al. 2011

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Glycerol 3-phosphate dehydrogenase (GPD) plays an important role in the energy metabolism and nutrition metabolism. In order to know about the biological functions of GPD of Clonorchis sinensis (C. sinensis), we identified a complete gene coding GPD from C. sinensis metacercaria cDNA library. This novel cDNA sequence contains 1,056 bp with a putative open reading frame of 351 amino acids and shares 74% identity with GPD from Schistosoma mansoni. Recombinant CsGPD was expressed and purified from Escherichia coli BL21 (DE3). Western blot analysis displayed that recombinant CsGPD can be recognized by anti-CsGPD serum and C. sinensis-infected serum. RT-PCR and immunolocalization analysis confirmed that GPD expressed both at the stage of adult worm and metacercaria of C. sinensis and immunolocated at the tegument of adult worm, tegument and tegumentary cells of metacercaria. Our current study has paved the way for the further researches about the biological functions involved in the growth of C. sinensis.

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Section: Methodsmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

Molecular cloning, expression, and immunolocalization of the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis

et al. 2011

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“…Adult worms and infected livers of rabbits were stained by immunofluorescence techniques as described by Yang et al (10). Adult worms and infected liver were fixed with 10% neutral formalin and embedded with paraffin and then sliced into sections.…”

Section: Sds-page and Western Blot Analysismentioning

confidence: 99%

Molecular cloning and tissue distribution of a Schistosoma japonicum gene encoding AMY-1

Tang³

et al. 2011

Mol Med Report

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Abstract.It is well known that the mammalian associate of Myc-1 (AMY-1) plays a significant role in spermatogenesis or cellular differentiation. A full-length complementary DNA (cDNA) encoding AMY-1 of Schistosoma japonicum (SjAMY-1) was identified and isolated from a cDNA library. The gene contained an open reading frame of 315 nucleotides, encoding 105 amino acids. Sequence analysis showed that SjAMY-1 shares 65.7% homology with Homo sapiens AMY-1 amino acids and contains a conserved domain from the AMY-1 family. In this study, we cloned and expressed a recombinant SjAMY-1 (rSjAMY-1) with a molecular size of 14 kDa. The native SjAMY-1 in soluble worm antigen was identified by anti-rSjAMY-1 sera in the Western blot analysis, which demonstrated the presence of this protein in the parasite. Immunofluorescence studies indicated a localization of SjAMY-1 in various tissues and organs including the tegument and subtegumental muscles in adult worms, the ventral sucker in cercariae and the internal structures of eggs. Given the key roles of mammalian AMY-1 in cell proliferation and differentiation, the characterization of SjAMY-1 may allow for a better understanding of the development of S. japonicum.

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“…6 In our previous study, one novel serpin named CsSERPIN was identified and characterized. 7,8 It is significantly highly expressed in metacercaria stage. The biochemical activities showed that rCsproSERPIN could significantly inhibit trypsin, chymotrypsin, and thrombin, indicating that it may play an important role in the information of metacercariae.…”

Section: Introductionmentioning

confidence: 99%

Comparison of two serpins ofClonorchis sinensisby bioinformatics, expression, and localization in metacercaria

Yang

Wang

et al. 2014

Pathogens and Global Health

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Clonorchiasis, which has been an important public health problem in China, is caused by ingestion of raw or undercooked fish contaminated by live metacercaria. Therefore, preventing fish from infecting is of great significance for controlling the disease. SERPINs (serine protease inhibitors) are well known as negative regulators of hemostasis, thrombolysis, and innate immune responses. In the present study, two full-length sequences encoding SERPIN were identified from metacercaria cDNA library of Clonorchis sinensis (C. sinensis) and were denominated as CsSERPIN and CsSERPIN3, respectively. Bioinformatics analysis showed that the two sequences shares 35.9% identity to each other. Both of the sequences have SERPIN domain and the greatest difference between the two domains is the reactive centre loop. Transmembrane region was found in CsSERPIN3 while not in CsSERPIN. The expression of the two CsSERPINs was significantly higher at the life stage of metacercaria than that of adult. The transcription levels of CsSERPIN and CsSERPIN3 at metacercaria stage were 3.249-and 11.314-fold of that at adult stage, respectively. Furthermore, the expression of CsSERPIN was 4.32-fold of that of CsSERPIN3 at metacercaria stage. Immunobiochemistry revealed that CsERPIN was dispersed at subtegument and oral sucker of metacercaria, while CsSERPIN3 localized intensely in the tegument of metacercaria of C. sinensis inside of the cyst wall. All these indicated that the CsSERPINs play important roles at metacercaria stage of the parasite. CsSERPIN may take part in regulation of endogenous serine proteinase and CsSERPIN3 may be involved in immune evasion and be a potential candidate for vaccine and drug target for clonorchiasis.

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Molecular cloning and characterization of a novel serpin gene of Clonorchis sinensis, highly expressed in the stage of metacercaria

Cited by 18 publications

References 17 publications

Molecular cloning, expression, and immunolocalization of the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis

Molecular cloning, expression, and immunolocalization of the NAD+-dependent glycerol 3-phosphate dehydrogenase (GPD) from Clonorchis sinensis

Molecular cloning and tissue distribution of a Schistosoma japonicum gene encoding AMY-1

Comparison of two serpins ofClonorchis sinensisby bioinformatics, expression, and localization in metacercaria

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