阅读说明：本技术 一种编码重组人骨形态发生蛋白质-2的氨基酸序列 (Amino acid sequence of coding recombinant human bone morphogenetic protein-2 ) 是由 曹建新 曾以申 于 2018-07-24 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种新的重组人骨形态发生蛋白质-2(rhBMP-2)的氨基酸序列及其编码的核苷酸序列,以及rhBMP-2的制备方法。本发明通过对rhBMP-2基因的核苷酸序列进行进一步优化,筛选出能够表达具有良好异位诱导成骨活性以及具有较好复性纯化效果的rhBMP-2的新的氨基酸序列。应用本发明所构建的工程菌能够诱导产生表达水平约为55%的重组rhBMP-2蛋白,所产生的目的蛋白相对于现有的重组rhBMP-2更易复性纯化,显示了出较好的复性纯化效果以及更高的诱导成骨活性。(The invention relates to the field of biotechnology, and discloses an amino acid sequence of a novel recombinant human bone morphogenetic protein-2 (rhBMP-2), a nucleotide sequence coded by the amino acid sequence and a preparation method of the rhBMP-2. The invention screens out a new amino acid sequence which can express the rhBMP-2 with good ectopic induction osteogenesis activity and better renaturation purification effect by further optimizing the nucleotide sequence of the rhBMP-2 gene. The engineering bacteria constructed by the invention can induce and generate recombinant rhBMP-2 protein with an expression level of about 55%, and the generated target protein is easier to renaturate and purify compared with the existing recombinant rhBMP-2, thereby showing better renaturation and purification effect and higher induced osteogenesis activity.)

1. An amino acid sequence for coding recombinant human bone morphogenetic protein-2, which is characterized in that the amino acid sequence is shown as SEQ ID No.1 or SEQ ID No. 2.

2. The amino acid sequence of claim 1, wherein the DNA sequence encoding said amino acid sequence is as shown in SEQ ID No. 3.

3. A method for preparing recombinant human bone morphogenetic protein-2, comprising the following steps:

(1) designing an amino acid sequence and a DNA sequence for coding the recombinant human bone morphogenetic protein-2;

(2) constructing an expression vector and transforming an escherichia coli host cell;

(3) screening positive clones for culture, and inducing the expression of target protein;

(4) renaturing and purifying the expression product to obtain the recombinant human bone morphogenetic protein-2.

4. The method according to claim 3, wherein the renaturation is carried out by dilution in the step (4), and the final concentration of the protein in the renaturation solution is controlled to be 0.05-0.5 mg/ml.

5. The preparation method according to claim 3, wherein the purification in step (4) is performed by multi-step ion exchange chromatography, and the specific steps are as follows:

(1) loading the recombinant human bone morphogenetic protein-2 solution with good renaturation on a strong anion column with good balance, and flushing the column with a balance buffer solution A to reach a baseline after loading;

(2) eluting with an elution buffer solution A in a gradient with gradually increased salt gradient, and collecting a main peak;

(3) mixing target peak solutions eluted and collected from the strong anion column, loading the mixed solution on the weak anion column, and flushing the mixed solution with a balance buffer solution B to reach a baseline after the loading is finished;

(4) elution was performed with a gradient of increasing salt gradient using elution buffer B and the main peak was collected.

6. The method of claim 5, wherein the equilibration buffer A comprises 10-50mM Tris-HCl, 1-5M urea, 1% -10% mannitol, pH 8.5-8.9; the elution buffer solution A comprises 10-50mM Tris-HCl, 1-5M urea, 1% -10% mannitol and 1-5M NaCl, and the pH value is 8.5-8.9; the equilibrium buffer solution B comprises 10-50mM phosphate buffer solution PB, 1-5M urea and 1% -10% mannitol, and the pH value is 6.0-6.5; the elution buffer solution B comprises 10-50mM phosphate buffer solution PB, 1-5M urea, 1% -10% mannitol and 1-5M NaCl, and the pH value is 6.0-6.5.

7. The amino acid sequence according to claim 1 or 2, which has been modified by glycosylation.

8. The amino acid sequence according to claim 7, wherein the glycosylation modification is modified by artificial in vitro modification or expression in eukaryotes.

9. The amino acid sequence of claim 8, wherein the artificial modification is a modification with PEG.

Technical Field

The invention relates to the field of biotechnology, in particular to an amino acid sequence of a novel recombinant human bone morphogenetic protein-2 (rhBMP-2), a nucleotide sequence coded by the amino acid sequence and a preparation method of the rhBMP-2.

Background

BMP-2 is an acidic glycoprotein containing 396 amino acid residues consisting of three parts, an N-terminal signal peptide, an intermediate propeptide and a C-terminal mature peptide, the mature peptide consisting of 114 amino acids, a part for inducing osteogenesis, which contains 7 cysteine residues, 6 of which form intramolecular disulfide bonds, and another part for forming intermolecular disulfide bonds, a heterologous biological activity of a monomer for forming an intermolecular mature peptide or a dimer thereof, wherein each monomer forms 3 pairs of disulfide bonds, and thus, the formation of a pair of glycosylation sites between two monomers is difficult to be studied, and the purity of the synthesized BMP-2 protein is not influenced by the activity of a glycoprotein, and the purity of the synthesized BMP-2 is not easily confirmed by a clinical study, and thus, the BMP- β is not influenced by a single factor of Transforming Growth Factor (TGF) - β superfamily, and thus, the BMP-2 is hardly influenced by a single factor of a bone and a skeletal defect repair, and spinal fusion, and is hardly influenced by a single factor of a bone origin of a bone and a vertebral bone defect, and is hardly influenced by a single factor of formation, i.2, and a factor of bone defect formation, which is indispensable for a bone repair, and a factor of a bone system formation, and a factor of formation, which is hardly formed by a C-2, which is hardly influenced by a single factor of a protein, a gene, a single factor of a single protein, a.

Currently, the preparation of rhBMP-2 is mainly carried out by two modes of eukaryotic expression vectors and prokaryotic expression vectors. Eukaryotic expression systems, such as CHO, COS and baculovirus systems, are frequently used abroad. Most eukaryotic expression products are secreted and are glycosylated, and the rhBMP-2 belongs to secreted protein and has glycosylation sites, so the eukaryotic expression system is an ideal expression system. The product prepared by the eukaryotic expression system has relatively high activity, but has the defects of low expression quantity, high production cost, long period, no contribution to large-scale production and the like. Currently, most of domestic and foreign rhBMP-2 is produced by a prokaryotic expression system using Escherichia coli as a host, and different long and short fragments of the rhBMP-2 mature peptide are usually obtained. Because the prokaryotic expression system does not have the process of post-translational processing and modification of polypeptide chains by eukaryotic cells, the eukaryotic gene protein cannot be correctly folded and post-translationally processed (such as disulfide bond formation, glycosylation, polymerization, protease degradation and the like), so that the biological activity and the utilization rate of the eukaryotic gene protein are restricted. Meanwhile, eukaryotic proteins expressed in prokaryotic expression systems usually aggregate in the cytoplasm of bacteria in an inactive manner to form insoluble inclusion bodies, and the natural activity of the eukaryotic proteins is recovered through treatments such as denaturation and renaturation. Chinese invention patent CN1215171C discloses a method for producing truncated recombinant human bone morphogenetic protein-2 mature peptide, wherein the truncated recombinant human bone morphogenetic protein-2 mature peptide is a nucleotide sequence artificially synthesized to code BMP-2C-terminal 107 amino acid residues, an initiation codon ATG is added in front of a first amino acid codon, a second amino acid arginine (R) is mutated into a lysine (K) residue, and the amino acid sequence is MKKLKSSCKR HPLYVDFSDV GWNDWIVAPP GYHAFYCHGE CPFPLADHLN STNHAIVQTLVNSVNSKIPK ACCVPTELSA ISMLYLDENE KVVLKNYQDM VVEGCGCR; the amount of the human truncated BMP-2 generated by the genetic engineering bacteria reaches 30-50% of the total soluble protein amount of the bacteria, and the human truncated BMP-2 has certain osteogenic activity. Chinese invention patent CN101787369B discloses an optimized recombinant human bone morphogenetic protein-2 DNA sequence and the preparation and application of its coded protein, the invention obtains rhBMP-2 with complete mature peptide (114 amino acid residues) by optimizing the DNA sequence, the product purity is over 95%, and the yield of purified dimer is about 21%. The prior several rhBMP-2 mature peptide fragments have relatively poor renaturation and purification effects on preparing rhBMP-2, and the biological activity of the final product is not high. One of the solutions to solve the above problems is to analyze the mature peptide gene by genetic engineering recombination technology, further optimize the nucleotide sequence of the target gene on the basis of not influencing and changing the molecular structure of the protein, and screen out a new amino acid sequence capable of expressing rhBMP-2 with good ectopic induced osteogenesis activity and good renaturation purification effect.

Disclosure of Invention

The invention aims to provide a novel amino acid sequence of recombinant human bone morphogenetic protein-2 (rhBMP-2), so that the expressed protein is easy to renaturate and purify, has better renaturation and purification effects and higher bioactivity and bone repair capacity.

In order to solve the technical problems, the technical scheme of the invention is as follows:

optimizing the amino acid sequence of rhBMP-2, and designing the amino acid sequence of the new recombinant human bone morphogenetic protein-2 as follows:

KRLK SSCKR HPLYV DFSDV GWNDW IVAPP GYHAF YCHGE CPFPL ADHLN STNHAIVQTL VNSVN SKIPK ACCVP TELSA ISMLY LDENE KVVLK NYQDMVVEGC GCR(SED ID No.1)。

furthermore, methionine M coded by initiation codon (AUG) is added into the N end of the protein sequence, and the amino acid sequence is as follows:

MKRLK SSCKR HPLYV DFSDV GWNDW IVAPP GYHAF YCHGE CPFPL ADHLN STNHAIVQTL VNSVN SKIPK ACCVP TELSA ISMLY LDENE KVVLK NYQDM VVEGC GCR(SED ID No.2)。

such amino acid sequences also include amino acid sequences modified by glycosylation, for example, amino acid sequences modified by PEG.

All rare codons were removed by codon optimization to become e.coli-preferred codons, whose optimized DNA sequence is shown below:

ATGAAACGTCTGAAAAGCAGCTGCAAACGTCACCCGCTGTACGTTGATTTCAGCGATGTTGGCTGGAACGATTGGATCGTTGCGCCGCCGGGCTACCACGCGTTCTACTGCCACGGCGAATGCCCGTTCCCGCTGGCGGATCACCTGAACAGCACCAACCACGCGATCGTTCAGACCCTGGTTAACAGCGTTAACAGCAAAATCCCGAAAGCGTGCTGCGTTCCGACCGAACTGTCTGCGATCTCAATGCTGTACCTGGATGAAAACGAAAAAGTTGTTCTGAAAAACTACCAGGATATGGTTGTTGAAGGTTGCGGTTGCCGTTAA(SED ID No.3)。

another object of the present invention is to provide a method for preparing recombinant human bone morphogenetic protein-2, comprising the steps of: (1) designing an amino acid sequence and a DNA sequence for coding the recombinant human bone morphogenetic protein-2; (2) constructing an expression vector and transforming an escherichia coli host cell; (3) screening positive clones for culture, and inducing the expression of target protein; (4) renaturing and purifying the expression product to obtain the recombinant human bone morphogenetic protein-2.

Preferably, the vector is expressed in eukaryotic cells, such as CHO cells, such that expression of the protein in eukaryotic cells is achieved with glycosylation modifications, and it is within the scope of the invention that the amino acid sequence is also active with glycosylation modifications. Alternatively, the protein may be modified artificially in vitro, for example, by using PEG, to greatly improve its stability. Our experiments found that the stability of the modified protein is improved by about 35-50% by comparing the activity of the protein expressed in vitro with the activity of the protein without modification.

Preferably, the renaturation is carried out by dilution in the step (4), and the final concentration of the protein in the renaturation solution is controlled to be 0.05-0.5 mg/ml.

Preferably, the purification is carried out by adopting multi-step ion exchange chromatography in the step (4), and the specific steps are as follows: (1) loading the recombinant human bone morphogenetic protein-2 solution with good renaturation on a strong anion column with good balance, and flushing the column with a balance buffer solution A to reach a baseline after loading; (2) eluting with an elution buffer solution A in a gradient with gradually increased salt gradient, and collecting a main peak; (3) mixing target peak solutions eluted and collected from the strong anion column, loading the mixed solution on the weak cation column, and flushing the mixed solution with an equilibrium buffer solution B to reach a baseline after the loading is finished; (4) elution was performed with a gradient of increasing salt gradient using elution buffer B and the main peak was collected.

Preferably, the equilibration buffer A comprises 10-50mM Tris-HCl, 1-5M urea, 1% -10% mannitol, and has a pH of 8.5-8.9; the elution buffer solution A comprises 10-50mM Tris-HCl, 1-5M urea, 1% -10% mannitol and 1-5M NaCl, and the pH value is 8.5-8.9; the equilibrium buffer solution B comprises 10-50mM phosphate buffer solution PB, 1-5M urea and 1% -10% mannitol, and the pH value is 6.0-6.5; the elution buffer solution B comprises 10-50mM phosphate buffer solution PB, 1-5M urea, 1% -10% mannitol and 1-5M NaCl, and the pH value is 6.0-6.5.

The invention screens out a new amino acid sequence which can express the rhBMP-2 with good ectopic induction osteogenesis activity and better renaturation purification effect by further optimizing the nucleotide sequence of the rhBMP-2 gene. The engineering bacteria constructed by the invention can induce to generate recombinant rhBMP-2 protein with an expression level of about 55 percent, the generated target protein is easier to renaturate and purify compared with the prior recombinant rhBMP-2, a better renaturation and purification effect is shown, the yield of the purified target protein is more than 60 percent or about 60 percent, and the purity is higher than 98 percent; and the expressed recombinant rhBMP-2 has higher induced osteogenesis activity and wide market prospect.

Drawings

FIG. 1 is a map of the pET28 plasmid used in the present invention.

FIG. 2 is the multiple cloning site of plasmid pET 28.

FIG. 3 is an SDS-PAGE electrophoretic analysis of rhBMP-2 induced expression.

FIG. 4 is a graph showing the result of SDS-PAGE electrophoretic analysis of renaturation, wherein 1: a molecular weight standard; 2-7: renatured rhBMP-2 dimer.

FIG. 5 is a diagram showing the result of SDS-PAGE analyzing purity, in which A is an electrophoretogram of a target peak collected by a strong anion column and B is an electrophoretogram of a target peak collected by a weak cation column, 1: a molecular weight standard; 2-10: and collecting the target peak.

FIG. 6 shows capillary electrophoresis purity analysis of rhBMP-2, detected by UV214 nm.

Fig. 7 is a new bone formed by radioactive examination and induction of ectopic osteogenesis experiments of rhBMP-2 mouse muscle pocket, wherein 1 is a diagram of induced osteogenesis effect of 108 peptide recombinant rhBMP-2, 2 is a diagram of induced osteogenesis effect of rhBMP-2 expressed by the amino acid sequence of the present invention, 3 is a diagram of induced osteogenesis effect of 109 peptide recombinant rhBMP-2, and 4 is a diagram of induced osteogenesis effect of 115 peptide recombinant rhBMP-2.

Detailed Description

The invention is further described below with reference to specific examples, which are set forth merely to illustrate how the invention may be carried out, but are not intended to limit the invention further. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

In the present invention, all percentages are weight/weight (w/w), unless otherwise specified, and all equipment and materials, etc. are commercially available or commonly used in the industry. Unless otherwise indicated, the examples employ methods that are within the ordinary skill in the art. Since the codon for methionine M (ATG) is the start codon for the translation of the protein, the specific example was conducted with the amino acid sequence SED ID No.2 containing methionine M at the N-terminus.