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人骨形态发生蛋白-2（hBMP-2, human Bone Morphogenetic Protein-2）是骨基质中的最重要骨生长因子和骨生成的启动因子，具有诱导骨髓基质细胞（BMSCs）分化增殖为骨组织的作用，可以成为骨修复基因治疗的理想目的基因。利用基因工程技术获得重组人BMP-2（rhBMP-2）可以实现珍惜的hBMP-2的高效表达。 本文以大肠杆菌（E.coli BL 21-DE3）为宿主细胞，pET-28a（+）质粒为载体表达人骨形态发生蛋白-2(hBMP-2)。摸索最佳培养条件后，实现了珍惜的hBMP-2的诱导表达，并进行了复性和诱导活性研究。 本实验从含BMP-2cDNA质粒及含pET-28a(+)质粒的BL 21(DE3) E.coli中分离出单菌落后扩大培养，提取含BMP-2全长基因的纯质粒，DNA琼脂糖凝胶电泳检验质粒的纯度。对其进行引物设计并在CDS段基因前后增加EoRⅠ和XhoⅠ酶切位点，胶回收得到纯度很高的BMP-2基因片段，与pMD18-T质粒连接。对pMD18-T-BMP-2基因片段及pET-28a(+)进行酶切，再用DNA连接酶酶连并复制，成熟的质粒转入感受态细胞BL 21(DE3) E.col 通过单因素实验摸索诱导温度、转速和诱导剂(IPTG)浓度对BMP-2表达量的影响。得出BMP-2优化诱导表达最佳条件为诱导温度25℃、转速200rpm和0.1mmol/L诱导剂浓度。 将表达的BMP-2经过含有His-tag标签的纯化树脂纯化后，纯化前后BMP-2纯度从0.422mg/ml提高到0.676mg/ml，提高了1.6倍。纯化后的的目的蛋白进一步复性，BMP-2经变性液溶解，装入透析袋内。尿素体系为6M，用复性缓冲液l梯度稀释样品，每30min 尿素浓度降低1 mol/L，透析温度为4℃。当复性体系中尿素浓度降至1 mol/L 时，置于4 ℃ 12 h，后再用复性液透析8 h，最后用50mmol/L 的磷酸钠缓冲液中透析8 h。其中4 h 换1 次液体，10 000 r/min 离心除去不溶物。 从SPF级wister大鼠股骨中提取BMSCs进行原代和传代培养，将第三代BMSCs与加入BMP-2前后的聚乳酸/羟基磷灰石（Poly L-Lactid /Hydroxyapatite, PLLA/HA）复合材料复合进行体外细胞培养，MTT法检测BMSCs在生物材料表面的增殖情况；SEM(扫描电镜)观察其表面超微机构及细胞在材料上的生长情况。ALP（碱性磷酸酶活性）检测细胞生长情况。结果表明：MTT结果显示，加入BMP-2前后的PLLA/HA多孔复合材料的的活细胞增殖的能力有一些提高，而且两者的活细胞增殖的能力都是随着时间的增加而增长的，这说明PLLA/HA这种材料可以作为细胞生长的载体材料，而且BMP-2具有增殖细胞的作用。添加了BMP-2之后的材料TiO2 500℃、TiO2+Fe3% 500℃、 硅500℃具有利更好的细胞增殖作用，MTT数据比未添加BMP-2的数据有明显提高，说明这三种材料骨髓间充质细胞生长无抑制作用，未发生细胞毒性反应，即有良好的生物相容性。 电镜照片中可以看出来，PLLA/HA，PLLA/BMP-2-HA对骨髓间充质细胞生长无抑制作用，未发生细胞毒性反应，细胞在材料表面能正常粘附、生长、增殖，均具有良好的细胞附着形态和细胞增殖率。 ALP结果显示，添加了BMP-2之后的材料具有利更好的细胞增殖作用，ALP数据比未添加BMP-2的数据有明显提高。实验结果证明BMP-2具有良好的生物诱导活性。 关键词 骨形态发生蛋白-2；表达纯化；优化；BMSCs；活性 |
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goodtimega
铁杆木虫 (著名写手)
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2楼2009-11-28 21:00:33
francisng(金币+5,VIP+0):依然感谢你 11-29 13:18
zap65535(金币-5,VIP+0):机器翻译不能给分 12-22 00:31
zap65535(金币-5,VIP+0):机器翻译不能给分 12-22 00:31
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Bone Morphogenetic Protein (hBMP human Bone Morphogenetic 2-2) Protein - Bone matrix is one of the most important Bone growth factors and Bone formation of induction, BMSCs Bone marrow stromal cells () function for Bone tissue proliferation and differentiation, can become Bone repair the ideal goal of gene therapy. Use of gene engineering technology for recombinant human BMP - 2 (2) rhBMP - can realize the value of hBMP - 2 to efficiently express. Based on escherichia coli (21 - DE3 E.c oli BL) for the host cell, pET 28a (+) - for the carrier plasmid expressing bone morphogenetic protein (hBMP - 2). The best condition, groping realized the treasure hBMP - 2 induced, and the refolding and induction activity. This experiment 2cDNA - including BMP from plasmid and pET 28a (+) - the plasmid DE3 21 (BL) E.c oli isolated colony expanded training, single extraction including BMP - 2 length of genes, pure plasmid DNA agarose gel electrophoresis tests plasmid purity. The design of primers and CDS gene Ⅰ EoR and increase Xho and Ⅰ s., plastic recycling enzyme obtained high purity of BMP - 2, and genetic fragments pMD18 - T plasmid connection. For pMD18 - T - BMP - 2 gene and pET 28a (+) - enzyme, DNA connection with enzymes enzyme and even copy, mature into normal cellular plasmid feeling DE3 21 (BL) E.c ol By single factor experiment, speed and temperature induced grope YouDaoJi (IPTG) concentration of BMP - 2 expression. That BMP - 2 optimization for induction induced optimum condition of 25 degrees Celsius temperature 200rpm speed, mmol/L and 0.1 YouDaoJi concentration. Will express BMP - 2 after His tag labeling containing purified resin - after purification, purification and BMP - 2 purity from 0.422 mg/ml to 0.676 mg/ml, improve the 1.6 times. After purification, the target protein further renaturation 2 - BMP via degeneration dissolving, load dialysis bag. Urea system with 6M, for the refolding buffer diluted sample, l gradient 30min urea concentration lower 1 mol/l for 4 degrees Celsius temperature, dialysis. When the refolding system of urea in concentration to 1 mol/L, on April 12 h c, reoccupy after 8 h, dialysis liquid utilized by the end 50mmol/L of sodium buffer dialysis 8 h. Four times liquid, h for 10 000 r/min centrifugal remove insoluble. From SPF wister rats were extracted from the femur BMSCs nestin cultivation, and third generation will BMSCs and join BMP - 2 after polylactic acid/Hydroxyapatite Poly (Lactid Hydroxyapatite/L - and PLLA/HA) composite material compound for in vitro cell culture, BMSCs MTT method in detecting the proliferation of biological material surface conditions, Scanning electron microscopy (SEM) to observe its surface ultrastructure and cells in the growth of the material. ALP (alkaline phosphatase activity) detection cell growth. Results: the results showed that the MTT before joining BMP - 2 PLLA/HA porous composite living cell proliferation, and improving the ability of both to live on cell proliferation capacity is as time increases, it shows PLLA/HA this material can be used as a carrier of the cell growth and BMP - 2 materials with proliferation cells. Add a BMP - 2 after TiO2 500 degrees Celsius, TiO2 materials Fe3%500 ° c + 5 ° c, silicon cell proliferation with better effect than did not add MTT data and the data is 2 - BMP, it can obviously improve the three kinds of materials of bone marrow stromal cells grow without inhibition, did not happen cells, namely toxicity of good biocompatibility. Electron micrograph can see, in PLLA/HA, PLLA/BMP - 2 - HA of bone marrow stromal cells grow without inhibition, without toxic reaction, cells in the cell surface can be normal growth, proliferation and adhesion, has the good cell proliferation rate and attached. ALP results show that adds BMP - 2 after the material with better effect of cell proliferation, ALP data than add BMP - 2 data has obviously improved. Experimental results prove that BMP - 2 with good biological induction activity. Keywords BMP - 2, Expression of purification, Optimization, BMSCs, active |
3楼2009-11-28 21:28:08