肝片吸虫Fh8明显增强STEC的Stx1可溶性表达及Stx1单克隆抗体制备

doi:10.7668/hbnxb.2016.05.007

摘要/Abstract

摘要： 志贺毒素1（Shiga toxin 1，Stx1），系五聚体蛋白，是产志贺毒素性大肠杆菌（Shiga Toxin-producing Escherichia coli，STEC）主要的毒素因子，但Stx1体外极不易可溶性表达，旨在克隆和表达志贺毒素1（Shiga toxin 1，Stx1）A亚基，体外获得可溶性表达的Stx1，以研制单克隆抗体。生物信息学软件分析Stx1A亚基氨基酸序列，Stx1A1亚基N端为信号肽段，其C端高疏水性和低免疫原性。PCR扩增stx1A亚基73~759的687个核苷酸，与肝片吸虫Fh8基因串联，克隆到低温表达载体pCold Ⅰ以构建重组菌，诱导表达重组毒素His-Fh8-Stx1A，SDS-PAGE分析蛋白表达形式。重组毒素His-Fh8-Stx1A免疫Balb/c小鼠，Sp2/0细胞融合筛选、制备阳性杂交瘤和单克隆抗体。PCR扩增Fh8和stx1a亚基并构建重组质粒pCold Ⅰ-His-Fh8-stx1，重组蛋白在原核细胞中得到高效表达，且以可溶性形式存在于菌体胞浆中。以重组His-Fh8-Stx1A为免疫原，成功制备3株能稳定传代并分泌Stx1的单克隆抗体（McAb）的杂交瘤细胞株，取其中1株成功制备高ELISA效价的腹水。Western Blot显示，纯化的单克隆抗体可与重组免疫原His-Stx1A和天然志贺毒素1发生特异性反应。本试验成功可溶性表达志贺毒素1A亚基，并获得多株单克隆抗体，为研制用于检测STEC的夹心ELISA和胶体金试纸条奠定理论基础。

关键词: 产志贺毒素大肠杆菌, 志贺毒素1, 肝片吸虫, Fh8, 可溶性表达, 单克隆抗体

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

中图分类号:

张雪寒, 张碧成, 张强, 汪伟, 何孔旺. 肝片吸虫Fh8明显增强STEC的Stx1可溶性表达及Stx1单克隆抗体制备[J]. 华北农学报, 2016, 31(5): 44-49. doi: 10.7668/hbnxb.2016.05.007.

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.

http://www.hbnxb.net/CN/Y2016/V31/I5/44

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