摘要
目的建立优化高效的碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、骨形态发生蛋白2(bone morphogenetic protein,BMP-2)蛋白重组方法,阐明二者在促成骨中的协同效应。方法优化bFGF和BMP-2成熟肽的基因序列密码子,获得适用于大肠杆菌Origami2体系表达的bFGF及BMP-2成熟肽cDNA片段;利用PCR法将bFGF成熟肽cDNA片段插入原核表达质粒pColdⅡ中,构建重组原核表达质粒pColdⅡ-bFGF;利用PCR法将BMP-2成熟肽cDNA片段插入原核表达质粒pET-30a(+)中,构建重组原核表达质粒pET-30a(+)-BMP-2,转入大肠杆菌Origami2中表达纯化;运用双酶切、质粒测序及Western blot技术证明Origami2表达体系的构建效果;进行细胞增殖实验、碱性磷酸酶(alkaline phosphates,ALP)染色观察和体内成骨效率检测等,探索bFGF、BMP-2在促成骨中的协同效应。结果双酶切和质粒测序鉴定证明成功构建重组大肠杆菌pColdⅡ-bFGF/Origami2和pET30a(+)-BMP-2/Origami2表达体系,Western blot检测发现蛋白正确表达。经过Ni-NTA亲和层析纯化后bFGF及BMP-2纯度高达90%。通过对二者比例的优化筛选,发现40 ng/mL bFGF+40 ng/mL rh BMP-2时,大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖效率、ALP活性及体内成骨效率最高,是二者促成骨的最优效组合。结论成功构建高效表达的pColdⅡ-bFGF/Origami2和pET30a(+)-BMP-2/Origami2表达体系,实现了bFGF及BMP-2的高效表达。bFGF和BMP-2的优化联合应用具有促成骨协同效应。
Objective To establish recombinant expression systems for basic fibroblast growth factor（bFGF） and bone morphogenetic protein（BMP-2） and investigate the synergetic effect of bFGF and BMP-2 in promoting osteogenesis. Methods The codons encoding mature peptide sequences of bFGF and BMP-2 gene were optimized and their cDNA fragments capable of expressions in E. coli Origami2 system were obtained. Using PCR method,the mature peptide cDNA fragments of bFGF and BMP-2 were separately inserted in the prokaryotic expression plasmids pCold Ⅱ and pET-30 a（＋） to construct the recombinant expression plasmids pColdⅡ-bFGF and pET-30 a（＋）-BMP-2,respectively. The plasmids were transferred into E. coli Origami2 for expression and the products were purified. Double enzyme digestion,sequencing analysis and Western blotting were used to verify the established Origami2 expression systems. The synergetic effect of the recombinant bFGF and BMP-2 proteins in promoting osteogenesis was tested by observing their effect on proliferation of rat one marrow mesenchymal stem cells（BMSCs）,ALP activity in mouse C2C12 myoblasts and osteogenesis of BMSCs in rat quadriceps femoris in vivo. Results Double enzyme digestion and sequencing analysis verified successful construction of recombinant E. coli pCold Ⅱ-bFGF/Origami2 and pET30 a（＋）-BMP-2/Origami2 expression systems. Western blotting demonstrated expressions of the two target proteins in the expression systems,and after purification with Ni-NTA affinity chromatography,the expressed bFGF and BMP-2 proteins reached a purity as high as 90%. At the optimized ratio,bFGF（40 ng/mL）combined with rh BMP2（40 ng/mL） obviously promoted the proliferation of rat BMSCs,enhanced ALP activity in C2C12 myoblasts,and efficiently promoted ectopic osteogenesis of BMSCs in rat quadriceps femoris. Conclusion We successfully constructed pCold Ⅱ-bFGF/Origami2 and pET30 a（＋）-BMP-2/Origami2 expression systems for efficient expression of bFGF and BMP-2. At the optimized ratio,bFGF and BMP-2 produce a synergistic effect to promote osteogenesis of BMSCs.
作者
涂兵
叶吉星
甘翼搏
张立泰
欧阳斌
罗向东
周强
TU Bing1 , YE Jixing1 , GAN Yibo1 , ZHANG Litai1, OUYANG Bin1 , LUO Xiangdong2, ZHOU Qiang1(1 Department of Orthopedics, National ＆ Regional United Engineering Laboratory of Tissue Engineering, 2Stace Key Laboratory of Trauma, Burns and Combined Injury, Institution of Burn Research, First Affiliated Hospital, Army Medical University （Third Military Medical University）, Chongqing, 400038, China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2018年第10期882-888,共7页
Journal of Third Military Medical University
基金
全军重点项目(BWS13C014-1)
西南医院军事医学创新计划(SWH2016JSZD-01)
第三军医大学军事医学预研基金(SWH2013JS04)~~
关键词
碱性成纤维细胞生长因子
骨形态发生蛋白2
基因重组
协同效应
成骨
basic fibrobIast growth factor
bone morphogenetic protein
genetic recombination
synergistic effect
osteogcnesis
