人酰基辅酶A:胆固醇酰基转移酶C端片段在大肠杆菌中的表达及其抗体的制备

Expression of human ACAT C-terminal fragment in E. coli and preparation of its antiserum

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。

Objective: To express ACAT C terminal fragment containing the membrane anchoring region 2 (ACATc) and prepare its antiserum. Methods: The gene encoding ACATc was cloned into vector pBLPAV7. Then the plasmid pBLPAV7 ACATc was transferred into E. coli strain AR68. Results: The expressed product of ACARc fused with BC domains of protein A (PABC ACATc) was detected and purified by the affinity chromatography with Sepharose 4B IgG column respectively. Conclusion: The antiserum against ACATc was prepared and can be used for the immunoassay of recombinant or natural ACAT.