摘要
目的克隆金黄色葡萄球菌肠毒素C2(SEC2)基因,添加亲和纯化标签进行原核表达,并对表达产物进行生物学活性分析。方法通过PCR从pGEM-T-SEC2质粒中亚克隆得到带有编码6个组氨酸的核苷酸序列的基因片断,构建了表达质粒pET-28a-SEC2,转化至大肠杆菌BL21中诱导表达。利用亲和层析纯化rSEC2,并采用MTT法对纯化后的rSEC2和天然SEC2生物学活性进行研究和比较。结果表达质粒pET-28a-SEC2通过测序,表明已连入正确表达目的蛋白的核苷酸序列。含有该质粒的表达菌株经IPTG诱导,产生约占菌体总蛋白40%的可溶性重组蛋白。SDS-PAGE显示,经Ni2+亲和柱纯化的目的蛋白达到了电泳纯。MTT法表明该rSEC2和天然SEC2均有显著的超抗原活性。结论成功构建了pET-28a-SEC2表达质粒,获得了与天然SEC2有着类似超抗原活性的高纯度rSEC2,为进一步研究该蛋白的抗肿瘤活性奠定了基础。
Objective To construct an E. coli expression system for the staphylococcal enterotoxin C2 （SEC2） with an affinity tag. Methods Amplified SEC2 gene with nucleotides coding for six hisfidines was subcloned from plasmid pGEM-T-SEC2 by PCR, an then the segment was inserted into pET-28a（ ＋ ） and an expression vector pET-28a-SEC2 was constructed. The protein was induced in E. coil BL21 with IPIG and purified by affinity chromatography. The bioactivity of both rSEC2 and native SEC2 were analyzed by MTT method. Results The confirmed nucleotides sequence could code the protein correctly, and the soluble rSEC2 was expressed efficiently in E. coLI BL21 with pET- 28a-SEC2. The protein purified by affinity chromatography resulted in one single band by SDS-PAGE detection. The superantigen activity of the recombinant protein was proved by lymphocyte proliferation test and tumor suppression test. Conclusion The expression plasmid pET-28a- SEC2 is constructed and the recombinant superantigen is expressed successfully, which may provide a foundation for the further research of the anticancer activity of SEC2.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第2期144-147,151,共5页
Immunological Journal
基金
浙江省科技厅重大科技攻关项目资助(2004C13041)
关键词
肠毒素
超抗原
克隆表达
组氨酸标签
亲和纯化
Enterotoxin
Superantigen
Recombinant expression
His-tag
Affinity chromatography
