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| Prokaryotic Expression, Purification and Preparation of Polyclonal Antibody for Wheat (Triticum aestivum) CWI-B1 |
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Abstract Cell wall invertase(CWI) is the key regulator which forms sugar concentration gradient between source and sink by catalyzing sucrose hydrolysis and promotes sucrose into sink. In order to uncover the important roles of CWI in wheat (Triticum aestivum), bioinformatics were utilized to analyse the protein properties and structure map of wheat TaCWI-B1. The prokaryotic expression vector pET28a-TaCWI-B1 was constructed by inserting the coding region of TaCWI-B1 into pET28a, and transformed into Escherichia coli strain BL21(DE3). The induction conditions, including isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, culture temperature and induction time, were optimized, and the recombinant protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified. After identifying the recombinant protein by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), the polyclonal antibody was prepared, its titer was measured by enzyme-linked immunosorbent assay (ELISA) and its specificity was analyzed by Western blot. The results showed that the full-length cDNA of TaCWI-B1 was 1 845 bp and this gene had a ORF of 1 776 bp and encoded a protein of 591 amino acid with molecular mass 65.92 kD and pI 9.26. The analysis based on software of Protscale and Swiss-model showed that TaCWI-B1 was a hydrophilic protein with a structure of N-acetyl glucosamine outside, and it was a major component of cell walls. The prokaryotic expression showed that TaCWI-B1 was expressed abundantly as inclusion bodies, and the optimum condition for the target protein production was 0.2 mmol/L of IPTG and induction at 28 ℃ for 6 h. The His-tag fused TaCWI-B1 protein was purified by Ni-NTA SefinoseTM Resin Kit and the MALDI-TOF-MS analysis showed that the similarity between recombinant protein and TaCWI-B1 was 99.992%, which indicated it was really the cell wall invertae of wheat. ELISA showed that the serum antibody titer of 2 TaCWI-B1 immunized rabbits reached over 1∶50 000. The result of Western blot demonstrated that the TaCWI-B1 polyclonal antibody could specifically recognize not only the recombined protein, but also the target protein from wheat. These results provide basic data for further investigation of TaCWI-B1 gene functions.
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Received: 05 January 2017
Published: 16 June 2017
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