|
|
|
| Analysis of Gene Evolution, Protein Expression and Identification of Echinococcus granulosus EgG1Y162 |
| TUERXUN Zulipiye1, CAO Chun-bao1, WEN Hao1, DING Jian-bing2, YIMITI Delixiati1,2 |
1. Base to Foster a State Key Lab for Major Disease Research in Xinjiang , The First Affiliated Hospital of Xinjiang Medical University, Urumuchi 830054, China;
2. College of Preclinical Medicine, Xinjiang Medical University, Urumuchi 830011, China |
|
|
|
Abstract Objective: To clone and identify Echinococcus granulosus egG1Y162 gene and analysis the protein expression, adaptive evolution and identification.of antigenicity. Methods: According to the gene sequence of emy162 , a pair of primers was designed from Echinococcus granulosus protoscolices, cystic wall students send layer, adults and eggs of four developmental stage respectively , extract the genomic DNA and total RNA, mRNA was reverse transcribed into cDNA,amplified egG1Y162 gene via PCR with genomic DNA and cDNA . Construct the recombinant plasmid of PUCm-T/EgG1Y162 and identified by PCR, enzyme digestion and sequencing to determine its correctness. The characteristics of EgG1Y162 gene were analyzed by DNAman software and MEGA4 software, and construct the phylogenetic tree of EgG1Y162 nucleic acid to further explore its homology. Fluorescence quantitative PCR detection egG1Y162 gene at four different developmental stages including Echinococcus granulosus protoscolices, cystic wall students expression layer, adults and eggs. The EgG1Y162 gene was cloned into prokaryotic expression plasmid PET-41a by using the directed cloning technology, and the positive clones were identified by the restriction analysis and PCR identification, and the sequence was determined by sequencing. IPTG was initially induced and expressed by EgG1Y162-GST recombinant proteins and identified by SDS-PAGE and Western blot assay. Results: EgG1Y162 gene were cloned from both two different developmental stages of Echinococcus granulosus, from total DNA clone obtained length for 1 680bp fragment , from a cDNA clone obtained length for 459bp fragment .The similarity of the EgG1Y162 gene was 91%, while the similarity of emY162 gene cDNA and EgG1Y162 was 95%. Further analysis showed that the sequence of EgG1Y162 gene was composed of 3 exons and 2 introns, the exon regions were 1~70,1064~1380and 1577~1648. The amino acids of 1~16 amino acids in the hydrophobic side constitute the EgG1Y162 signal peptide sequence, and 35~115 amino acids form a large fibronectin splicing FN3133~152 amino acids which constitute the carboxyl terminal transmembrane region. The result of DNA sequencing showed that the length of EgG1Y162 gene was 360bp and 120 amino acids were encoded.. By fluorescence quantitative PCR detection found egG1Y162 in adults, germinal layer, protoscolex and the egg stage had different degrees of expression. But the majority egG1Y162 expression in the adult stage, at most a relative value is 19.526 difference was statistically significant (P< 0.01), secondly germinal layer, 5.122, again in protoscolex stage, relative value 5.083, in the egg stage at least for 1.6588. The prokaryotic expression plasmid of PET-41a/EgG1Y162 was induced by SDS-PAGE, and the IPTG assay showed that the recombinant protein of EgG1Y162-GST was successfully expressed and expressed in the relative molecular weight of 44KDa. Western blot analysis showed positive blot bands, the molecular weight of 44kda, egG1Y162 recombinant protein can with Echinococcus granulosus infection after 40 days of canine sera react; serum from patients with hydatid disease also have positive reaction. Conclusion: Successfully cloned egG1Y162 antigen gene, sequence comparison analysis showed high similarity with cDNA of the egG1Y162 cDNA and emy162, differences in gene mainly exists in the intron region, the egG1Y162 antigen gene is a new gene. The expression of EgG1Y162 in adult was the most. The recombinant protein was successfully induced, and the recombinant protein of EgG1Y162 has significant antigenicity.
|
|
Received: 16 September 2015
Published: 06 January 2016
|
|
|
|
|
| [1] Johannes E, Peter D. Biological, epidemiological and clinical aspects of Echinococcosis, a zoonosis of increasing concern.Clinical Microbiology Reviews, 2007,17(1):107-135.
[2] Wu J H. Border areas hydatid disease causes and prevention of epidemic response. Medical Information, 2007,20(4):682-683.
[3] Jiang C P. Current epidemic status of Echinococcosis in chin . Endemic Diseases Bulletin,2002,17(3):77-79.
[4] Wen H, Xu M. Practical Echinococcosis. Beijing: Science Publishing House, 2007. 1-19.
[5] Katoh Y, Kouguchi H, Matsumoto J,et al. Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis. Biochim Biophys Acta,2007,1780(1):1-6.
[6] Katoh Y, Kouguchi H, Matsumoto J, et al. The vaccination potential of EMY162 antigen against Echinococcus multilocularis infection. Biochim Biophys Acta, 2007,363(4): 915-920.
[7] Zeng S H, Wang S P,Zhang J X, et al. Analyzing the structures and function of SJCWL06 encoding protein of Schistosoma japonicum by bioinformatics.Int J Med Parasit Dis, 2006,33(1):10-14.
[8] Zhang W B,Li J, MeManus D P, et al. Concepts in immunology and diagnosis of hydatid disease. Clinieal Mierobiology Review,2003,16(l):18-36.
[9] Eekert J, Sehantz P,Gasser R, et al.Geographic Distribution and Prevalenee. In:Eekert J,Gemmell M A,Meslin F X, ed.WHOI/OIE Manual on Eehinoeoceosis in Humans and Animals:a Public health problem of global concern.Paris:World Organization for Animal Health,2001.100-141.
[10] Eekerta J, Conrathsb F,Taekmannb K, et al. Eehinocoeeosis: an emerging or reemerging zoonosis?International Journal for Parasitology, 2000, 30 (13): 1283- 1294.
[11] Heath, D D. The use of a Recombinant Antigen to Immunize Sheep against Echinococcus granulosus, Proceedings of the International Congress of Hydatidology. Beijing, 1993,10- 13.
[12] Lightowlers M W. Vaccination against cestode parasites. Int J Parasitol, 1996, 26 (8/9):819-824.
[13] Lightowlers M W, Jensen O, Fernandez E, et al. Vaccination trials in Australia and Argentina confirm the effectiveness of the Eg95 hydatid vaccine in sheep. Int J Parasitol,1999,29(4):531-534.
[14] Akira I, Yasuhito S, Hiroshi Y, et al. Development of Em18-immunoblot and Em18-ELISA for specific diagnosis of alveolar Echinococcosis.Acta Tropica,2003,8 (5): 173-182.
[15] Gabriela A,Santiago M,Di P, et al. Binding properties of Echinococcus granulosus fatty acid binding protein. Biochimica et Biophysica, 2001,15(33): 293-302.
[16] 曹春宝,丁剑冰.棘球蚴感染中的免疫逃避相关分子.中国病原生物学杂志,2008,3(10):791-792. Cao C B, Ding J B. Molecules related with immune escape in echinococcosis.Journal of Pathogen Biology,2008,3(10):791-792.
[17] Li H Z, Graham R,Bernd H, et al. Cloning and expression of a cDNA encoding a nonintegrin laminin-binding protein from Echinococcus granulosus with localization of the laminin-binding domain. Molecular and Biochemical Parasitology,1997,8 (7) 183-192.
[18] Virginio V, Hernández A,Rott M,et al.A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease. Clin Exp Immunol,2003,13(9):309-315.
[19] Lightowler M, Gauci G, Chow C, et al. Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites. International Journal for Parasitology, 2003,3 (3): 1207-1217.
[20] Cecilia C,Ramiro M,Andrea R, et al. Production and characterization of monoclonal antibodies against excretory/secretory products of adult Echinococcus granulosus, and their application to coproantigen detection. Parasitology International, 2005,5 (4):43-49. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
