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目的 :为了表达人ACATC端片段并制备其抗体。方法 :将编码人ACAT羧基端包含第二跨膜(463 550氨基酸残基)的cDNA片断克隆至不同的大肠杆菌表达载体中 ,构建了系列表达质粒 ,并在多种大肠杆菌中进行了表达研究。结果 :在详细探索表达条件的基础上 ,经温敏诱导 ,成功地在大肠杆菌AR68中表达了N端融合ProteinABCdomain的人ACAT羧基末端包含第二个跨膜区的片段 ,表达量约20mg/L菌液。通过优化纯化条件 ,经Sepharose4B IgG亲和层析柱分离纯化得到表达产物 ,并制备获得其阳性抗血清。结论 :这些工作为进一步表达完整的ACAT蛋白、研究ACAT基因表达调控及其结构与功能的关系奠定了基础。
Purpose: To express human ACATC-terminal fragments and prepare their antibodies. METHODS: cDNA fragment containing the second transmembrane (463-550 amino acid residues) encoding the carboxyl terminus of human ACAT was cloned into different E.coli expression vectors, and a series of expression plasmids were constructed and expressed in various E.coli . Results: Based on the detailed exploration of the expression conditions, a second transmembrane region containing the second transmembrane region was successfully expressed in Escherichia coli AR68, and the expression level was about 20 mg / L Bacteria liquid. By optimizing the purification conditions, the expressed product was purified by Sepharose 4B affinity chromatography and the positive antiserum was obtained. Conclusion: These results laid the foundation for the further expression of intact ACAT protein and the study of the relationship between ACAT gene expression and its structure and function.