Fibulin-1蛋白的真核表达与ELISA检测方法的建立

Establishment of ELISA for measurement of Fibulin-1 employing eukaryotic protein expression

目的建立检测血清中Fibulin-1蛋白浓度的ELISA方法。方法构建Fibulin-1蛋白真核表达载体，以人Fibulin-1基因编码序列为模板，将目的片段定向克隆入载体并转入毕赤酵母菌，进行Fibulin-1蛋白的真核表达。表达产物纯化后，Westernblotting检测证明纯化的重组蛋白的抗原特异性。以该蛋白为定标绘制标准曲线，建立人血清ELISA检测方法并检测其精密度和回收率，最后初步分析人血清中Fibulin-1的含量。结果从真核表达体系获得了纯化的Fibulin-1蛋白，其具有良好的抗原特异性；建立的ELISA方法线性相关系数W=0．98，线性范围为12．5—800ng／mL，检测灵敏度为6．25ng／mL，蛋白回收率在94．33％～109．33％之间，CV均〈10％，具有良好的重复性；检测30例正常人血清样本中Fibulin-1的含量约为（31±8）μg／mL。结论建立的ELISA方法可用于检测血清中的Fibulin-1蛋白含量。

Objective To establish an ELISA method for Fibulin-1 detection in human serum. Methods Firstly, to construct an eukaryotic expression vector of fibulin-1 and express the fusion protein, The human fibulin-1 gene coding sequence was used as templates to amplify the target DNA fragments, which were direct-cloned into a Pichia vector. Secondly, the recombinant fibulin-1 protein was expressed and purified after induction, and western blotting was used to test the antigenieity and specificity of the purified recombinant protein. Then the protein was used as standard to generate an ELISA standard curve, and the accuracy and protein recovery rate were analyzed. Finally, this ELISA method was used to determine the protein concentration of fibulin-1 in human serum. Results The recombinant fibulin-1 protein was successfully expressed from the eukaryotic expression system and purified. The purified protein had good antigenicity and specificity. The linear correlation coefficient of this ELISA method was 0.98, the linear determination range was 12.5-800 ng/mL, the testing sensitivity was 6.25 ng/mL, the protein recovery rate was from 94.33% to 109.33%, the variation coefficient （CV）was less than 10%. The concentration of Fibulin-1 in 30 serum specimens was determined by the this ELISA method and the result was （31±8）μg/mL.