摘要
目的:从苦瓜籽中克隆HIV-1整合酶抑制剂MAP30,构建其表达载体,并在大肠杆菌中进行表达,用于MAP30在细胞内的转运机理研究。方法:从成熟苦瓜种子中提取基因组DNA,通过PCR的方法扩增出MAP30基因的全长,并将其克隆进入原核表达载体pET-30a,转化Eoli.BL21(DE3),IPTG诱导表达,Ni-NTA亲和纯化,梯度咪唑洗脱。MTT法分析生物学活性。结果:测序发现插入的MAP30基因序列正确,SDS-PAGE鉴定表达产物分子量正确,且MAP30主要以可溶形式表达。MTT法测定重组的MAP30具有生物学活性,且对肿瘤细胞的毒性远远大于对正常细胞的毒性。结论:本研究成功地构建了MAP30的表达系统,并实现了MAP30的可溶性表达,表达后的蛋白具有生物学活性,可用于抗体-毒素的肿瘤靶向药物的合成。
Objective： To clone and produce ribosome inactivating protein MAP30 from the seeds of Momordica charantia L（bitter melon）,and to evaluate the biological activity of the recombinant protein.Methods： The DNA sequence encoding MAP30 was cloned from the fresh seeds of Momordica charantia by PCR,the target DNA fragments were sequenced after T-A cloning.The expression plasmid was constructed by inserting the MAP30 fragment into vector pET30a.MAP30 was expressed in E.coli by addition of IPTG into final concentration of 1.0 mmol/L.The recombinant MAP30 was identified by SDS-PAGE,and the biological activity of MAP30 protein was evaluated by using MTT assay in cancer cells and normal cells following fluid-phase endocytosis.Results： The nucleotide and amino acid sequences of the cloned MAP30 were identical with those of reported MAP30.The solubility of recombinant protein was analyzed by SDS-PAGE,and the MAP30 was mainly produced in soluble form.The recombinant MAP30 showed a greater cytotoxicity to cancer cells than that to normal cells.Conclusion： The gene of MAP30 has been successfully cloned.The recombinant MAP30 protein expressed by E.coli is bioactive.
出处
《浙江大学学报（医学版）》
CAS
CSCD
北大核心
2010年第3期264-271,共8页
Journal of Zhejiang University(Medical Sciences)
基金
浙江省重大科技专项(编号2009C13041)
国家自然基金(批准号30670424)
浙江大学医学院中青年科研启动基金
