蝎镇痛抗菌活性肽BmK AS的融合表达及蛋白切割  

Expression of analgesic-antimicrobial peptide BmK AS from scorpion in fusion form and recombinant protein hydrolysis

作　　者：杨东萍[1] 崔勇[2] 张敬武 杜秉娜[1] 王月秋[1] 张景海[1] 

机构地区：[1]沈阳药科大学生命科学与生物制药学院,辽宁沈阳110016 [2]沈阳药科大学医疗器械学院,辽宁本溪117004 [3]大连市药品检验所,辽宁大连116021

出　　处：《沈阳药科大学学报》2015年第2期148-153,共6页Journal of Shenyang Pharmaceutical University

基　　金：国家自然科学基金资助项目(81473432)

摘　　要：目的构建镇痛抗菌活性肽BmK AS原核融合表达载体,实现可溶性表达,获得重组活性肽BmK AS。方法利用本实验室已构建成功的pET 28a-AS质粒,设计引物经过聚合酶链式反应,将目的基因克隆至pET 32a载体中,构建融合表达载体pET 32a-AS,优化诱导表达条件,实现BmK AS在大肠杆菌BL21(DE 3)中的融合可溶性表达,通过金属离子螯合亲和薄层色谱法对重组蛋白进行初步分离纯化,获得重组融合蛋白后采用凝血酶进行切割去除纯化标签,经SDS-PAGE检测切割效果。结果成功构建重组表达质粒,优化得到低温诱导促进蛋白可溶性表达的方法;采用金属离子螯合亲和薄层色谱法获得重组蛋白;电泳结果表明凝血酶可以切割融合蛋白。结论实现活性肽BmK AS在大肠杆菌中的融合可溶性表达,经凝血酶切割后获得重组镇痛抗菌活性肽BmK AS,为后续药理活性研究奠定基础。Objective To construct a prokaryotic expression vector of BmK AS with analgesic-antimicrobial activity,and the recombinant active peptide BmK AS was obtained by soluble expression.Methods The plasmid of pET28a-AS constructed in our laboratory was used for the template.According to the sequence of BmK AS,the primers were designed.The recombinant fusion expression vector pET32a-AS was obtained by PCR.The c DNA of BmK AS previously cloned was inserted into pET32 a.After optimizing the condition of expression,the fusion protein was expressed in soluble form.The metal chelating affinity chromatography method was used to purify the recombinant protein.The recombinant fusion protein without purification tag cut by thrombin could be obtained,and the cutting efficiency was confirmed by SDS-PAGE.Results a.The recombinant plasmid pET32a-AS was successfully constructed.The recombinant protein was mostly expressed in soluble form,with methods of low temperature induction to promote the protein expression; b.The recombinant protein was purified by metal chelating affinity chromatography; c.The results of SDS-PAGE showed that thrombin could cut fusion protein.Conclusions The active peptide BmK AS is successfully expressed in soluble form in E.coli.After cutting by thrombin,the recombinant analgesic-antimicrobial active peptide BmK AS has been obtained,these will lay the foundation for subsequent pharmacological activity research.

关 键 词：镇痛抗菌活性 可溶性表达 融合 分离 纯化 凝血酶 

分 类 号：R963[医药卫生—微生物与生化药学]