梅花鹿骨钙素的原核表达与活性研究  

Prokaryotic expression and activity of sika deer osteocalcin

作　　者：张楠楠[1] 李宏灵 李帅军[1] 张鹏飞[1] 赵雨[1] 曲毅[1,2] ZHANG Nannan LI Hongling LI Shuaijunl ZHANG Pengfei ZHAO Yu QU Yi(Development Center of Traditional Chinese Medicine and Bioengineering, Changchun University of Chinese Medicine,Changchun 130117,China Jilin Food and Drug Safety Monitoring Center,Changchun 130033,China)

机构地区：[1]长春中医药大学中医药与生物工程研究开发中心,长春130117 [2]吉林省食品药品安全监测中心,长春130033

出　　处：《吉林中医药》2017年第1期62-66,共5页Jilin Journal of Chinese Medicine

基　　金：吉林省科技发展项目(20140622003JC);吉林省科技发展计划项目(20130101142JC)

摘　　要：目的利用基因工程技术表达纯化梅花鹿骨钙素重组蛋白(Bone gla protein,BGP)并初步研究其活性,为研究梅花鹿茸生长发育提供技术支撑。方法通过PCR技术获取BGP基因,构建p GEX-BGP-His表达载体,并转化至Rosseta(DE3)宿主菌中,IPTG诱导表达。经Ni Sephrose 6 Fast Flow分离纯化,SDS-PAGE鉴定纯化产物。MTT法分析BGP对鹿茸软骨细胞chon-sv40的生物活性。结果经双酶切、测序证实p GEX-BGP-His表达载体构建成功;成功诱导表达相对分子量33.5 KDa的重组蛋白;以37℃,0.1 mmol/L IPTG诱导5 h条件下重组蛋白表达量较高;蛋白纯化后纯度可达95%;重组蛋白对鹿茸软骨细胞chon-sv40具有明显促增殖作用。结论骨钙素与鹿茸软骨细胞chon-sv40的增殖密切相关,为后续BGP在骨代谢机制以及鹿茸有效成分的研究奠定基础,对鹿茸生长机制的研究具有重要意义。Objective By gene engineering the recombinant protein of sika deer bone gla protein （Bone gla protein, BGP） was expressed, purified and preliminary studied on its activity, which provided technical support for the development of antler growth. Methods The BGP gene was obtained by PCR technique, and the pGEX-BGP- His expression vector was constructed and transformed into Rosseta （DE3） host strain, and induced with IPTG to express the recombinant protein. Isolation and purification of Ni Sephrose 6 Fast Flow and identified by SDS-PAGE. The biological activities of BGP fusion protein on antler chondrocytes chon-sv40 were analyzed using the methods of MTT. Results Confirmed by double enzyme digestion and sequencing that the expression vector of pGEX-BGP- His was constructed successfully; A 33.5 KDa protein was induced and obtained; The higher expression level of the recombinant protein was noted at 0.1 mmol/L IPTG after incubation at 37 ℃ for 5h; The protein purity up to 95% after purification; The recombinant protein has obvious effect on proliferation of antler chondrocytes chon-sv40. Conclusion Osteocalcin and antler chondrocytes chon-sv40 proliferation is closely related,to lay the foundation for the follow-up study of BGP in bone metabolism and the effective composition of pilose antler, it has significant means for the study of the mechanism of antler growth.

关 键 词：梅花鹿 骨钙素 原核表达 鹿茸软骨细胞 

分 类 号：R282.74[医药卫生—中药学]