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Recombinant expression of calcium-binding protein from Clonorchis sinensis and preliminary evaluation of its application in immunodiagnosis

PU Jue-biao1,2，TANG Li-li3，YIN Cen-nan1，DU Xin-yue1, WU Jian-hua1, ZHAO Wei1, #br# LIU Deng-yu3, WANG Zhao-jun1, WU Chen-yun1*   

  1. 1 Department of Immunology and Microbiology, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China; 2 Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China; 3 Department of Parasitology, School of Preclinical Medicine, Guangxi Medical University, Nanning 530021, China
  • Online:2018-06-30 Published:2018-07-02

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Abstract:

Objective To clone, express the calcium-binding EF-hand-domain-containing protein of Clonorchis sinensis (Cs16) by using the prokaryotic and eukaryotic expression systems, and evaluate its application in immunodiagnosis. Methods The Cs16 gene was amplified by PCR using specific primers and cDNA library of Clonorchis sinensis, and sub-cloned into prokaryotic and eukaryotic expression vectors pGEX-4T-1 and pPIC9K, respectively. The recombinant vectors pGEX-4T-1-Cs16 and pPIC9K-Cs16 were transformed into Escherichia coli BL21 and yeast GS115 for protein expression. The recombinant proteins were purified and analyzed for purity and antigenicity by SDS-PAGE and Western blotting, respectively. The purified recombinant protein was used as the coating antigen for indirect ELISA to evaluate its diagnostic performance against serum samples from 24 clonorchiosis patients and its cross-reactivity to serum samples from schistosomiasis patients.  Results PCR resulted in a 515 bp of Cs16 fragment. The recombinant plasmid pGEX-4t-1-Cs16 and pPIC9K-Cs16 were verified by enzymatic digestion and sequencing. SDS-PAGE analysis showed that the two recombinant plasmids were solubly expressed in E. coli BL21 and yeast GS115, producing recombinant protein Cs16 with Mr of 16 000. Western blotting results showed that the recombinant proteins were specifically recognized by anti-GST and anti-His. ELISA showed that the sensitivity of recombinant Cs16 proteins expressed in the prokaryotic and eukaryotic expression systems was 70.8%(17/24) and 54.2%(13/24), respectively, and the specificity was 95.2% (20/21) and 100% (21/21), respectively, with significant differences between the two systems (P　<　0.05). The cross-reactivity with sera from schistosomiasis patients was 1/10.  Conclusion The recombinant Cs16 can be expressed prokaryotically and eukaryotically, and has potential applications in immunodiagnosis of clonorchiasis.
  

Key words: Clonorchis sinensis, Calcium binding protein, Prokaryotic expression, Eukaryotic expression, Immunodiagnosis