基于特异性蛋白Gαa的比浊法检测β-葡聚糖

doi:10.11944/j.issn.1000-0518.2016.06.150325

摘要/Abstract

摘要：

通过基因重组技术,克隆表达了β-葡聚糖特异性结合美洲鲎G因子α亚基片段a(Gαa)蛋白,并建立了β-葡聚糖比浊检测方法。克隆的Gαa基因相对分子质量为40000(1251 bp),浓度为2 g/L,纯度达到98%以上。该蛋白能与βG特异性结合,紫外分光光度计在340 nm下检测的吸光度与βG含量呈正线性相关,线性范围3.125~200 mg/L,R²=0.997,检测限3.125 mg/L。结合力实验表明,该蛋白与βG具有良好的亲和性及特异性。该方法具有成本低、快速、专一性强等特点。

关键词: 美洲鲎G因子α亚基片段a特异性蛋白, β-葡聚糖, 比浊法

Abstract:

In this paper, a beta-glucan binding protein of horseshoe crab(limulus polyphemus) factor G-subunit α fragment-a was cloned, expressed and purified. A turbidimetry assay for the detection of beta-glucan was established. The DNA fragment of the binding protein gene was cloned into an expressional plasmid pET-15b. The protein was expressed with 6x-Histag on N-terminal and purified with Ni-column. The purified protein has relative molecular mass of 40000(1251 bp) on SDS-PAGE and the concentration is 2 g/L with the purity of >98%. The highly purified protein can combine with beta-glucan specifically with an affinity constant K_D of 3.14×10^-9 mol/L. The absorbance measurement at 340 nm using the ultraviolet spectrophotometer shows that the absorbance is linearly correlated with the concentration of beta-glucan. The linear range is 3.125~200 mg/L, R² is 0.997, and the detection limit is 3.125 mg/L. The novel turbidimetry assay for the detection of beta-glucan has low cost, rapid, and highly sensitive and specific.

Key words: horseshoe crab factor G-subunit αfragment-a binding protein, beta-glucan, turbidimetry assay

田振华, 张昊, 于源华. 基于特异性蛋白Gαa的比浊法检测β-葡聚糖[J]. 应用化学, 2016, 33(6): 727-732.

TIAN Zhenhua, ZHANG Hao, YU Yuanhua. Turbidimetry Assay for Detection of beta-Glucan by Gαa Binding Protein[J]. Chinese Journal of Applied Chemistry, 2016, 33(6): 727-732.

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