Polypeptide of recombinant human bone morphogenetic protein-2
a morphogenetic protein and polypeptide technology, applied in the field of biotechnology, can solve the problems of difficult to meet clinical needs, low bone bmp-2 level, limited fresh human bone source, etc., and achieve good ectopic osteoinduction activity, good renaturation and purification effect, and easy denature and purification
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example 1
Design of Novel Amino Acid Sequence of rhBMP-2 and Construction of Recombinant rhBMP-2 Expression Vector
[0026]1. In One Embodiment, the Polypeptide of rhBMP-2 has the Amino Acid Sequence as Follows:
(SEQ ID NO: 1)KRLK SSCKR HPLYV DFSDV GWNDW IVAPP GYHAF YCHGECPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSAISMLY LDENE KVVLK NYQDM VVEGC GCR.
[0027]Further, the N-terminal of the above amino acid sequence is added with methionine M encoded by the initiation codon (ATG). The amino acid sequence is as follows:
(SEQ ID NO: 2)MKRLK SSCKR HPLYV DFSDV GWNDW IVAPP GYHAF YCHGECPFPL ADHLN STNHA IVQTL VNSVN SKIPK ACCVP TELSAISMLY LDENE KVVLK NYQDM VVEGC GCR.
[0028]Further, by optimizing the codons, all rare codons are removed to become E. coli-preferred codons, at the same time, the restriction site BamHI (GGATCC) is added at the terminal. The optimized DNA sequences are as follows:
(SEQ ID NO: 3)ATGAAACGTCTGAAAAGCAGCTGCAAACGTCACCCGCTGTACGTTGATTTCAGCGATGTTGGCTGGAACGATTGGATCGTTGCGCCGCCGGGCTACCACGCGTTCTA...
example 3
In Vitro Activity Assay of Recombinant rhBMP-2 in C2C12 Cells
[0056]C2C12 cells (mesenchymal stem cells) were cultured in DMEM high glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin and 100 g / mL streptomycin) under the conditions of 37° C. and 5% CO2 and digested with 0.25% trypsin for passage every 1-2d. C2C12 cells were inoculated in a 24-well plate, and when cell fusion reached around 30%, recombinant rhBMP-2 was added for continuous culture. After 5 days of culture, the medium was discarded and washed twice with pre-cooled PBS buffer. After lysed with lysate for 5 min, the supernatant was centrifuged and the activity of alkaline phosphatase (ALP) was quantitatively determined according to the instructions of the kit.
[0057]Controlled Trial:
[0058]In order to compare the biological activities of recombinant rhBMP-2 having the novel amino acid sequences of the present invention, three kinds of reported polypeptides were used for controlled trial. The engineered b...
example 4
Effect of Three Vectors on Mouse Muscle Ectopic Osteogenesis Induced by Recombinant RhBMP-2
[0068]The recombinant rhBMP-2 aqueous solution was mixed with high-concentration gelatin, chitosan or natural coral, and the recombinant rhBMP-2 was uniformly combined with the three vectors to form a composite material by vacuumizing. Each vector contained 100 μg rhBMP-2, which was lyophilized and disinfected by ethylene oxide for later use.
[0069]Six normal ICR male mice were randomly divided into three groups, two mice in each group. After anesthesia with 1% sodium pentobarbital, the hind limbs were depilated and disinfected, and the skin was cut to separate the spatium intermusculare, and then implanted with a certain amount of the above composite material. Among them, the implanted materials in the first group used high concentration gelatin as a vector, and chitosan was used as a vector in the second group, and natural coral was used as a vector in the third group. Mice were given antibio...
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