کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2089685 1545914 2016 8 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی بیوتکنولوژی یا زیست‌فناوری
پیش نمایش صفحه اول مقاله
Characterization of a single-chain variable fragment specific to Cronobacter spp. from hybridoma based on outer membrane protein A
چکیده انگلیسی


• The protein sequence of OmpA of Cronobacter spp. were analyzed.
• A scFv based on protein OmpA specific to Cronobacter spp. was constructed.
• The whole preparation process of scFvH81 only required five days.

Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as antigen. The protein sequences of OmpA of Cronobacter spp. were analyzed first. The results showed protein OmpA with length of 347 amino acids was conserved in Cronobacter genus (94.83%–100% of protein identity) and was greater than that observed for the other genera tested (8.28–91.64% of protein identity). Then, purified protein OmpA expressed in E. coli was used to prepare hybridoma and to construct scFv further. The scFv was named scFvH81 and analyzed by bioinformatics. The model of scFvH81 built by homologous modeling had a good quality (residues in disallowed regions: 3%) and showed that scFvH81 had a standard pocket-like site. Purified scFvH81 was prepared by denaturation and renaturation of inclusion body and it showed a good specificity and its affinity of Ka = 2.39 × 106 M− 1. Therefore, it could be used in the detection and the pathogenesis study of Cronobacter spp.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Microbiological Methods - Volume 129, October 2016, Pages 136–143
نویسندگان
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