人血清淀粉样蛋白SAA1蛋白的原核重组表达  

Recombination and Expression of SAA1 Fusion Protein

作　　者：郝雪微[1] 马翠[2] 吴静红 李金桐[3] 陈硕[4] 

机构地区：[1]哈尔滨医科大学大庆校区检验学院生物化学教研室,黑龙江大庆163000 [2]哈尔滨医科大学检验学院免疫教研室,黑龙江大庆163000 [3]哈尔滨医科大学大庆校区检验学院,黑龙江大庆163000 [4]哈尔滨医科大学大庆校区药学院生物制药教研室,黑龙江大庆163000

出　　处：《现代生物医学进展》2016年第25期4829-4833,共5页Progress in Modern Biomedicine

基　　金：国家自然科学基金项目(81400353);黑龙江省卫生厅科技项目(2013-120)

摘　　要：目的:重组及表达人血清淀粉样蛋白SAA1融合蛋白,为进一步制备早期诊断冠心病的单克隆抗体做准备。方法:应用PCR技术,以成人肝脏的c DNA文库做模板,扩增出长度为315 bp的人血清淀粉样蛋白SAA1基因,并将其分别与带有HIS标签的PET-32a载体及GST标签的PGEX-4T-1载体连接,转化入DH5α感受态细胞中进行克隆并测序鉴定。并在大肠杆菌表达菌BL21菌中表达SAA1融合蛋白,之后分别应用SDS-PAGE及Western blot技术检验重组蛋白的纯度。结果:经SDS-PAGE凝胶电泳后,我们分别看到了分子量为32k D和分子量为38k D的两个蛋白条带,这两个条带分别为PET-32a-SAA1重组蛋白和PGEX-4T-1-SAA1重组蛋白,且这两种蛋白在超声后存在于菌液的沉淀中,证明在大肠杆菌中实现了不溶性的包涵体形式的表达,经Western blot鉴定,分别以HIS标签抗体和GST标签抗体为第一抗体,以鼠二抗为第二抗体,证明所表达的蛋白质为SAA1融合蛋白。结论:采用原核表达方法,经免疫纯化可获得高纯度的SAA1融合蛋白,为进一步制备相应的单克隆抗体和开发以SAA1增高为指标之一的冠心病诊断试剂盒打下基础,为冠心病的诊断开辟新途径。Objective： To recombine and express a recombinant, Serum amyloid A（SAA1） fusion protein, in E. coli and further prepare its monoclonal antibody which used for early diagnostic method of coronary artery heart disease. Methods： The gene encoding SAA1 was cloned using PCR techniques from c DNA of Adult liver, the amplified gene was inserted into vectors p ET-32 a with HIS tag and p GEX-4T-1 with GST tag, and was subcloned into DH-5α competent cells. The recombinant plasmids were constructed and transformed into E.coli BL21 cells, and then the protein expression of SSA1 with His-Tag and GST-Tag expression（including His-Tag and GST-Tag, respectively） were induced by IPTG and identified by SDS-PAGE and Western blotting. Results： A band of DNA about315 bp by DNA sequencing was confirmed as SAA1 gene, the expression system was constructed, and soluble expression of SAA1 fusion protein was achieved successfully, which was identified by Western blotting. Conclusions： Fusion protein SAA1 with high purity could be expressed in prokaryotic system. The study does a further preparation of its monoclonal antibody and lays a foundation for opening up new ways for early diagnosis of coronary artery diseases.

关 键 词：血清淀粉样蛋白 原核重组 冠心病 

分 类 号：R-33[医药卫生] R541.4