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Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

Mol Med Rep. 2017; 
NiWei-Wei,HuangWen,WuDe-Qin,ZhouYan-Jun,JiChun-Mei,CaoMeng-Da,GuoMiao,SunJin-Lu,WeiJ
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DNA Sequencing Following solubilization of the inclusion bodies using 8 M urea, the supernatant was loaded onto a Nickel column [GenScript (Nanjing) Co., Ltd.], washed with running buffer containing 20 mM Tris-HCl, 100 mM NaH2PO4, 10 mM imidazole and 8 M urea (pH 8.0), and eluted with elution buffer containing 20 mM Tris-HCl, 100 mM NaH2PO4, 250 mM imidazole and 8 M urea (pH 8.0). The nucleic acid sequence of mature Pla a 1 was synthesized by GenScript (Nanjing) Co., Ltd. (Nanjing, China) and was subcloned into a pET-28a vector (Novagen; Merck KGaA, Darmstadt, Germany) using EcoRI and XhoI sites, and the clone was verified by Sanger DNA sequencing, as described previously (21). Get A Quote

摘要

Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the fo... More

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