The Production Of Recombinant Human Bone Morphogenetic Protein 2 And 4 The Changes Of Bone Morphogenetic Protein 2 Expression In The Limb Bones Of The Tail-suspended Rats

Bone Morphogenetic Proteins ( BMPs ) are a kind of secretive proteins that include more than 20 members. Except for BMPl they all belong to TGF- β superfamily. BMPs have many important physiological functions. During the development of embryo they take an important part in inducing the formation of bone system, hematopoietic system, kidney, liver and neural system. In the stage of adult they can induce the stem cells in these systems to develop to the corresponding mature cells to repair the damnifications of these systems.BMP2 and BMP4 are two important members of BMP family, they have 86% homologous amino acid sequence and many similar physiological functions. BMP2 is a differential factor which induces the formation of osteoblasts so it plays a key role in the regeneration of bone when it has been damaged. Just because of this function, BMP2 isvery prospective in clinic use for speeding the healing of bone fracture, curing bone nonunion and bone default. As an inducing factor it has been used in the bone tissue engineering that makes it plays more important role in treating bone fracture and bone default.BMP4 can induce the ectopic formation of cartilages or bones but what makes it prospective in future clinical use are its important role in the development of the hematopoietic system and a potential as a hematopoietic growth factor. In cooperate with GM-CSF and EPO, BMP4 increases the number of both erythroid and granulocyte/ monocyte colonies formed in semi-solid medium, which is not shown in other BMPs. BMP4 can also induce neural stem cells to differentiate to mature nerve cell and this function may bring breakthrough in the diseases of neural system.In a word, BMP2 and BMP4 are very prospective in clinical use. There are many researches on preparing BMP for clinic using earlier. At first BMPs were extracted from fresh bones but the extracted protein had many problems such as low yield, poor purity, bad reproducibility and heterogeneous immunogenicity. The development of molecular cloning started a new way for BMP preparation. In genetic engineering, there are two kinds of expression system update, eukaryotes and prokaryotes. Eukaryotic expression system can give products with good activity. But it has common problems such as low production, difficult purification and high costs that make the products too costly to be widely used in clinic. Expressing BMP2/4 in Eschericha coli can solve such problems. However, eukaryotic proteinsynthesized in prokaryotic cell can not process to form acitive products and must be renatured in a proper way before use. The protein renaruration in vitro is always a troublesome matter and each protein has different renaturation means. BMP2/4 is a dimmer connected by one disulfide bond and each monomer has three intertwined disulfide bridges known as cystine knots. Renatuation of BMP2/4 seems more difficult than other proteins because its renaturation should make all these disulfide bonds connected correctly. There had been many reports on renaturation of BMP2, but how to raise the production of renatured active BMP2 and how to get soluble renatured BMP2 still remained puzzled.BMP4 has similar structure with BMP2, but there are rare reports on its large-scale expression , purification, renaturation and bioactivity testing. On the other hand there has been no new gene construction strategy for BMP4 to facilitate its renaturation.Bone loss in space flight threatens astronauts' lives. It has been thought that the mechanism of space bone loss is bone formation suppression. BMP2 is a key factor inducing the formation of osteoblasts. The expression of BMP2 is suppressed during space flight and it is one of the reasons causing space bone loss or BMP2 can prevent this bone loss hasn't been discussed before.This research focuses on producing active recombinant human bone mophogenetic protein 2/4 mature peptides in large scale. 1. To explore the influences of different conditions on the renaturationof rhBMP2m such as concentration of protein, concentration ofurea, pH, temperature, and volume of the renaturation dialysis buffer and find a better way for rhBMP2m renaturation by using a dialysis method.2. To clone the cDNA coding for human bone morphogenetic protein 4 mature peptide (hBMP4m) and insert the gene to the temperature induced vector pDH2 to build the expression vector pDH2-B4m then express the peptide in Escherichia coll At last to explore the fermentation, purification and renaturation technology for BMP4 and test the bioactivity of renatured protein.3. Design and construct rhBMP4m duplex (rdhBMP4m4m) gene and express it in Escherichia coli to make foundation for its further fermentation, purification, renaturation and bioactivity test.4. To compare the differences of the physical characters such as length, diameter, wet weight, dry weight, ash weight of the limb bones and mandible between the control group rats and the tail-suspension group rats. And compare the BMP2 expression in those bones by immunohistochemistry stain.After research we have got some results:1. For the first time we successfully renature rhBMP2m with high protein concentration in high concentration urea and got the best renaturation condition. The yield of rhBMP2m dimmer was the highest in following conditions: high protein concentration (4mg/ml) , high urea concentration (6M) of renaturation dialysis buffer, 250 fold volume of outer renaturation dialysis buffer, low temperature (10°C) and acidic renaturation dialysis buffer. Therenatured protein accounted for 56% of total protein. For the first time we got the soluble renatured rhBMP2m at pH4.8 that can be filtrated by 0.2 u m membrane to eliminate bacteria and the renatured protein showed good bioactivity by inducing the expression of reporter gene at ng/ml scale in vitro and inducing ectopic bone formation at 1 mg in the thigh of Kun-ming mouse.2. BMP4 was expressed by using a temperature-inducible system in Escherichia coli. The engineering bacteria strain that stably expressed rhBMP4m after 50 generations was built. A further mass expression by high-density ferment method was conducted. The OD600 value of the culture reached 46 at the end of the fermentation. The expressed insoluble rhBMP4m was then purified first by inclusive body washing. Then, the protein dissolved in urea submitted to ion chromatography and obtained 7.13 g rhBMP4m with 98% purity from 8 liters fermentation liquid. The purified rhBMP4m was further renatured by dialysis method to form the active dimers. The biological activity of the renaturated rhBMP4m was confirmed by its ability to induce ectopic formation of cartilage and bones in the quadriceps femoris of adult mice in 3 weeks and its ability to promote the expression of reporter gene in C2C12 cell culture line.3. Constructed the temperature inducible vector containing rdhBMP4m4m gene and got high expression in Escherichia coli.4. BMP2 expressed in femur and tibia was significantly lower in tail suspended rats than in control rats. Some physical characters such...