Fasciola hepatica 8 Significantly Enhances Soluble Expression of Stx1 from STEC and Stx1 Monoclonal Antibody Preparation

doi:10.7668/hbnxb.2016.05.007

Abstract

Abstract: Shiga toxin 1(Stx1),AB5 pentamers protein,is one of major virulent factors of Shiga Toxin-producing Escherichia coli (STEC).The N-terminal fragment of Stx1A subunit encodes a signal peptide,and its C-terminal amino acid sequences presents high hydrophobicity and low immunogenicity by bioinformatics software analysis,and these features lead to expression failure and inclusion of target protein.In this study,our aim is to clone stx1 gene and express soluble protein for development of monoclonal antibodies against Stx1A.The 210 bp of Fh8 gene from Fasciola hepatica was cloned into pCold Ⅰ with Nde Ⅰ and Xho Ⅰ,and the middle fragment between 73-759 ntof stx1A subunit was amplified,fused with Fh8 gene to construct recombinant strain of BL21(DE3)/pCold Ⅰ-Fh8-stx1. His-Fh8-stx1A was immunized Balb/c mice to prepare positive hybridomas and monoclonal antibodies by Sp2/0 cell fusion screening.We successfully constructed a recombinant plasmid pCold Ⅰ-Fh8-stx1a,expressed His-Fh8-Stx1A in prokaryotic cells in soluble form.Three stably secreting anti-Stx1A monoclonal antibodies(McAb) of hybridoma cell lines were developed,one of them was used to prepare high titer ascites.Western Blot assay showed ascites after purified are able to react with recombinant His-Fh8-Stx1A and Shiga toxin 1 crude extract.We successfully prepared soluble Stx1A protein with good antigenicity,and three monoclonal antibodies with high titers,these will lay substantial foundation for future study,i.e.developing sandwich ELISA and colloidal gold test strip to detect STEC.

Key words: Shiga toxin-producing Escherichia coli, Shiga toxin 1, Fasciola hepatica, Fh8, Soluble expression, Monoclonal antibody

CLC Number:

ZHANG Xuehan, ZHANG Bicheng, ZHANG Qiang, WANG Wei, HE Kongwang. Fasciola hepatica 8 Significantly Enhances Soluble Expression of Stx1 from STEC and Stx1 Monoclonal Antibody Preparation[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(5): 44-49. doi: 10.7668/hbnxb.2016.05.007.

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