雄激素含量测定用蛋白微阵列的制备

利用分子生物学技术对鼠雄激素受体结合域(rARLBD)重组表达,结合新兴的蛋白质微阵列技术建立了一种快速无污染的检测方法,用于临床雄激素检测及新药发现。实验将编码ARLBD的cDNA片段(888bp)插入到含有多聚组氨酸标签的表达载体pET32a中构建了表达质粒pET32a/AR,并将其转化到大肠杆菌BL21中。经IPTG低温诱导,得到高效表达的可溶性ARLBD融合蛋白产物,并通过NiNTA凝胶亲和吸附纯化。将纯化得到的ARLBD使用芯片点样仪固定到硅烷化-多糖表面片基,制备得到ARLBD蛋白质微阵列。实验得到的特异性结合曲线表明在微阵列上的受体蛋白能够保持功能构像。通过Scatchard方程计算得到微阵列上ARLBD与荧光标记的睾酮结合的Kd值为5.32nmol/L。浓度依赖性标准曲线表明这一方法有较高的灵敏度,最低检测限达1pmol/L。应用这一方法对224例健康中国老年人血清雄激素水平进行了测定,研究证明了该方法的可靠性,并首次提供了一定样本量的我国老年人血清活性雄激素水平的参考值。这一技术的建立将为生物工程技术产品与临床检测和分子水平化合物筛选有效地结合提供一条新的途径。

Preparation of Protein Microarrays for Androgen Determination

The purpose was to express soluble protein of the rat androgen receptor ligand binding domain (rAR-LBD) in Escherichia coli, and to develop a rapid and non-hazardous method based on protein mircoarrays technology for the clinical androgen determination and new drugs discovery. The cDNA segment coding rAR-LBD (888bp) was used to produce expressing vector pET32a/AR with vector pET32a. The plasmid was transformed into E. coli strain BL21 for expression, followed by IPIG induction. Large amount of fusion protein containing His-Tag linked to the amino terminal region of the receptor was produced in soluble form. After affinity purification by a Ni-NTA affinity resin, the AR-LBD protein was used to prepare protein microarrays. The receptor protein was printed to propyltrimethoxysilane-polysaccharide-coated slides to make protein microarrays by the array printer. From the results of the affinity tests, the dose-dependence curve shape suggested a positive cooperative binding of androgen with AR-LBD microarrays. After Scatchard analysis, the affinity constant (K_d) of AR-LBD bound to testosterone was 5.32nmol/L. The standard curve showed that this method has a good correlation (r2=0.98), and the sensitivity can reach 1pmol/L. To test the reliability of the AR-LBD microarrays, this method was used to determinate the androgen level of serum samples. 224 serum samples from healthy Chinese aging men and women were collected and investigated. Based on the data, this method can give a reliable result, and the reference values of serum androgen level in aging Chinese were provided for the first time. In conclusion, the development of the LBD protein microarrays can be an effective way to make good use of bio-technological product in clinical determination and new drugs discovery.