抗菌肽CM与血管内皮生长因子VEGF_(121)在大肠杆菌中表达及鉴定  

Expression and identification of fusion protein of cecropin A-melittin and VEGF_(121) in E. coli

作　　者：王会岩[1] 尹海燕[2] 尹翌秋[1] 刘孝菊[1] 赵宏鑫[1] 秦玉侠[1] 刘敏[1] 李校堃[1] 

机构地区：[1]吉林农业大学生物反应器与药物开发教育部工程研究中心,吉林长春130118 [2]长春工程学院机电学院

出　　处：《吉林大学学报（医学版）》2010年第2期285-290,共6页Journal of Jilin University：Medicine Edition

基　　金：吉林省科技厅科技发展计划项目资助课题(20090244);吉林省教育厅"十一五"科学技术研究项目资助课题(20070309)

摘　　要：目的:构建和表达人VEGF121的基因工程菌,为肿瘤血管的靶向治疗提供实验数据。方法:通过PCR引物延伸的方法,获得SUMO-CM-VEGF121融合基因;将该融合基因与原核表达载体pET22b连接后转化至Rosetta-gami(DE3)宿主细胞中,通过IPTG诱导获得可溶性表达。将融合蛋白经DEAE阴离子交换层析、Ni-NTA和分子筛等方法进行纯化,用SUMO酶切除分子伴侣SUMO后,最终获得融合蛋白CM-VEGF121。结果:DNA测序,设计合成的SUMO-CM-VEGF121融合基因为774bp;所构建的表达菌株Rosetta-gami(DE3)/pET22b-SUMO-CM-VEGF121在20℃诱导24h可溶性最好,其可溶性表达为菌体可溶性蛋白的21%。Westernblotting检测该表达产物与人VEGF单克隆抗体具有特异性结合能力。结论:原核表达载体pET22b和Rosetta-gami宿主菌为CM-VEGF121最合适的条件,解决了其表达量低和不可溶问题。Objective To construct and express a gene engineer bacteria which expresses human VEGF121 and provide a experimental foundation for targeting therapy of tumor vessel.Methods SUMO-CM-VEGF121 fusion genes were obtained by PCR.The VEGF121 was fused with SUMO-CM by PCR,and the fused gene was transformed into E.coli Rosetta-gami（DE3）.Soluble proteins were obtained at high level by IPTG.The fused protein was purified by DEAE sepharose and Ni-NTA affinity chromatography.Once cleaved from SUMO,the purity of CM-VEGF121 was obtained.Results The acquired gene fragments of SUMO-CM-VEGF121 were identified by digestion and DNA sequencing,and the fusion gene was 774 bp;the best condition for soluble protein was to cultivate the bacteria of Rosetta-gami（DE3）/pET22b-SUMO-CM-VEGF121 at 20℃ for 24 h,with a soluble expression level at 21%.Western blotting result showed that this protein had the same immunogenicity with human VEGF antibody.Conclusion The prokaryotic expression vector pET22b and host bacterium Roestta-gami are most suitable for CM-VEGF121,which solves the problem of low expression and insolubility.

关 键 词：抗菌肽CM 血管内皮生长因子 融合蛋白 纯化 

分 类 号：Q78[生物学—分子生物学]