梅花鹿VEGF的原核表达与纯化  

Prokaryotic Expression and Purification of VEGF from Sika Deer (Cervus nippon)

作　　者：胡薇[1] 孙宝龙[1] 张蕾[1] 张宇鹏[1] 高双娜 刘蓝月 韩香玉[1] 

机构地区：[1]吉林农业大学生命科学学院,长春130118

出　　处：《农业生物技术学报》2017年第7期1139-1145,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31572372和No.30972083);国家级大学生创新创业训练计划项目(No.201510193034)

摘　　要：鹿茸是一个天然的细胞生长因子库,鹿体最活跃的部位是鹿茸顶端组织,里面含有大量的生长因子。这些生长因子可为鹿茸的临床作用研究提供生物化学基础,多种生长因子对鹿茸的增殖具有重要的促进作用。为了获得具有生物学活性的蛋白质,本研究将梅花鹿(Cervus nippon)血管内皮生长因子(vascular endothelial growth factor,VEGF)成熟肽基因与p ET-30a连接构建VEGF基因原核表达载体,鉴定成功后,将p ET-30a-VEGF转入Rosetta大肠杆菌(Escherichia coli)感受态细胞进行诱导表达(0.6 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG),20℃,16 h),并对表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDSPAGE)检测。目的蛋白通过Ni-Agarose柱试剂盒进行纯化,通过复性以获得生物学活性。再应用咪唑蓝(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT)法检测重组蛋白对小鼠成纤维细胞(mouse embryonic fibroblast cell line,NIH3T3)的影响。鉴定结果显示,成功构建原核表达质粒;SDSPAGE电泳结果表明,在Rosetta中成功检测到融合蛋白的表达;MTT法结果显示,不同浓度的重组蛋白均能促进NIH3T3的增殖,并且随着浓度的增加,细胞的增殖速度也随之加快;其中,蛋白添加量为200 ng/m L、孵育48 h时,增殖效果较为明显。研究结果将为深入探讨VEGF在鹿茸快速增殖过程中的功能和调控机理提供实验依据。Velvet is a natural cell growth factor library. The top tissue of the antler as the most active growth point of the deer body to store a large number of growth factors, they are the biochemical basis of the clinical role of deer antler. A variety of growth factors have important roles in promoting the proliferation of antler. The rapid growth of velvet and the generation of blood vessels in cartilage tissue are inseparable and vascular endothelial growth factor （VEGF） can promote blood cell formation and promote the formation of blood vessels. Therefore, the study of sika deer （Cervus nippon） VEGF will provide a reference for the mechanism of rapid growth of velvet. To obtain the biologically active protein, the pET-30a prokaryotic expression vector and pMD- 18T-VEGF were digested with Hind ~[ and BamH I. The digested product was recovered separately. VEGF mature peptide gene from Cervus nippon was inserted to expression vector pET-30a by T~ DNA ligase to obtain recombinant plasmid. The recombinant plasmids were identified by PCR and identified by double digestion. The correct recombinant plasmid was made into glycerol. Proteins were expressed （0.6 mmol/L isopropyl [3-D-1-thiogalactopyranoside （IPTG） for 16 h at 20 ~C） in the host Rosetta cells in this study. The results were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE）. Target protein was purified by Ni-Agarose affinity column and renatured to obtain the biologically active protein. Target protein on the proliferation of mouse embryonic fibroblast cell line （NIH3T3） cell was analysed by 3-（4, 5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide （MTT）. A specific band of about 519 bp was obtained by double digestion of pET-30a-VEGF with 2 restriction endonucleases BamH I and Hind 1]I, which were consistent with the expected results. The PCR results of pET-30a-VEGF were used as templates to show a band corresponding to the size of the target band. The results showed that the VEGF gene of s

关 键 词：梅花鹿 血管内皮生长因子(VEGF) 原核表达 蛋白纯化 小鼠成纤维细胞(NIH3T3) 

分 类 号：S825[农业科学—畜牧学]