鹅胆固醇7α-羟化酶基因克隆及原核表达

摘要

To further study the regulatory mechanisms of Cholesterol 7a-hydroxylase (CYP7A1) in bile acid synthesis and cholesterol metabolism, the complete cDNA of CYP7A1 gene(2 279 bp) was cloned by RT-PCR and RACE from goose (Anser anser) liver. The CDS of CYP7A1 gene was sub-cloned into pET-28a to construct pET-28a-cyp7αl plasmid. The recombinant plasmid pET-28a-cyp7αl was transformed into E. coli BL21 (DE3) and the recombinant protein expres-sion was induced by IPTG. The recombinant protein His-CYP7A1 was purified with His-Bind Purification Kit. The recombinant protein was immunized into the Bal b/c mice to obtain the polyclonal antibodies. Antigenic of His-CYP7A1 and the specificity of mouse anti-goose CYP7A1 polyclonal antibodies were detected by Western blotting. The amino acid sequence of goose CYP7A1 protein has high homology with that of chicken(93%), rat(66%), human(67%), mo-nodelphis domestica(68%) and mouse(66%). The SDS-PAGE analysis result of His-CYP7A1 showed that the recombinant protein molecular size was about 59 ku, consistent with the predict-tion. High-purity His-CYP7A1 protein was obtained through His-tag affinity purification. The mouse anti-goose CYP7A1 polyclonal antibodies were obtained with high sensitivity (1 : 104). The mouse anti-his and anti-goose CYP7A1 antibodies could recognize antigens of His-CYP7A1 by Western blotting with mouse anti-goose CYP7A1 polyclonal antibodies with better specificity.The current results contribute to further understanding of gene structure and function of CYP7A1 and provide an important molecular tool for the functional study of CYP7A1.%为进一步研究胆固醇7α-羟化酶(CYP7A1)在胆汁酸合成和胆固醇代谢过程中的调节机制,采用RT-PCR和RACE方法从鹅(Anser anser)肝组织克隆CYP7A1基因全长cDNA序列,亚克隆其CDS区并构建原核表达载体pET-28a-cyp7α1.重组质粒在大肠杆菌BL21(DE3)菌株中经IPTG诱导表达.经His-Bind纯化试剂盒纯化的His-CYP7A1免疫Bal b/c小鼠,制备小鼠抗鹅CYP7A1多克隆抗体,Western blotting验证His-CYP7A1免疫原性和抗体的特异性.序列分析结果表明,该蛋白与鸡、大鼠、人、短尾猊、小鼠的CYP7A1蛋白氨基酸序列同源性分别为93 ％、66％、67％、68％、66％.SDS-PAGE分析结果表明,重组蛋白His-CYP7A1得到正确高效表达,该His0CYP7A1分子质量约为59 ku,经纯化获得高纯度重组His-CYP7A1蛋白.制备了小鼠抗鹅CYP7A1多克隆抗体,效价达1∶104.Western blotting检测表明,该蛋白能够被小鼠抗His抗体和小鼠抗CYP7A1抗体所识别,小鼠抗CYP7A1多克隆抗体具有较好的特异性.试验结果为进一步研究CYP7A1基因结构和CYP7A1在鹅肝脏脂类代谢中的作用提供了基础和技术手段.