基于特异性蛋白Gαa的比浊法检测β-葡聚糖  

Turbidimetry Assay for Detection of beta-Glucan by Gαa Binding Protein

作　　者：田振华[1] 张昊[1] 于源华[1] 

机构地区：[1]长春理工大学生命科学与技术学院,长春130022

出　　处：《应用化学》2016年第6期727-732,共6页Chinese Journal of Applied Chemistry

基　　金：吉林省科技厅资助项目(20140309013YY)资助~~

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。In this paper,a beta-glucan binding protein of horseshoe crab（ limulus polyphemus） factor Gsubunit 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 p ET-15 b. 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 KDof 3. 14 × 10- 9mol / 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.

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 

分 类 号：O652[理学—分析化学] R446[理学—化学]