CN114075569A - Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application thereof - Google Patents

Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application thereof Download PDF

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CN114075569A
CN114075569A CN202111363112.XA CN202111363112A CN114075569A CN 114075569 A CN114075569 A CN 114075569A CN 202111363112 A CN202111363112 A CN 202111363112A CN 114075569 A CN114075569 A CN 114075569A
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gdnf
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bdnf
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赵永强
甘振磊
俞君英
张颖
胡青松
焦璐琰
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Nuwacell Ltd
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Abstract

The invention provides a method for expressing recombinant neurotrophic factor fusion protein, the recombinant neurotrophic factor fusion protein and application thereof, belonging to the technical field of functional proteins. The invention obtains the high biological activity dimeric GDNF/BDNF recombinant protein by constructing a high expression vector and screening a high expression cell strain by using a cell expression system, and solves the defects of low expression level, high price, low biological activity and the like of the recombinant GDNF/BDNF on the market. The obtained GDNF/BDNF has very important significance for the basic research and clinical grade cell culture in the aspects of subsequent nerve growth and development and the like.

Description

Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application thereof
Technical Field
The invention belongs to the technical field of functional proteins, and particularly relates to a method for expressing recombinant neurotrophic factor fusion protein, the recombinant neurotrophic factor fusion protein and application thereof.
Background
Lin, L F et al, 1993, isolated and cloned a neurotrophic factor, named Glial Derived Neurotrophic Factor (GDNF), from the discovery of rat glioma cell line B49 cells and screening of human gene libraries with oligonucleotide probes (Lin LT et al, 1993). GDNF is a nerve cell trophic factor having important biological functions secreted from glial cells in brain tissue, belongs to a member of TGF-beta superfamily, has the effect of promoting the growth and differentiation of neurons, shows potential neurotrophic effects on dopaminergic neurons and motor neurons (Henderson et al, 1994), and also has important effects in participating in programmed death of nerve cells, promoting survival of neurons, participating in repair of axonal injury, and the like (Wang Rong Liang, 2004).
The function of GDNF has been extensively studied at home and abroad, and GDNF in the embryonic stage can promote the development of the nervous system, and in the adult stage, GDNF can repair neuroinflammation, injury and degenerative change. Sun M et al used herpes simplex virus vectors to express GDNF and found that GDNF had significant behavior-correcting effects on mice when it was injected into brain striatum in a targeted manner in mouse animal models of Parkinson's disease (Sun M, et al, 2005). Studies on the unilateral lenticular nucleus-infused GDNF in parkinson patients showed significant, persistent bilateral symptom-improvement with no significant adverse reactions (Slevin JT et al, 2006).
GDNF is a homodimeric secreted protein containing disulfide bonds. The preprotein is a signal peptide sequence containing 18 amino acid residues, and the mature protein after protein processing contains 134 amino acid residues and contains two glycosylation sites. The molecular weight of the protein dimer with biological activity is about 32-42 kD, the molecular weight of the monomer is 18-22 kD, and the biological activity is lower. Since GDNF is present in a low amount in tissues and cannot be obtained in large amounts by biological extraction, it is particularly important to obtain GDNF having biological activity by genetic engineering methods. The protein expression comprises a prokaryotic expression system and a eukaryotic expression system, wherein the prokaryotic expression system is simple to operate, can contain large gene segments and has low production cost, but the eukaryotic expression system can better keep the biological activity of the protein and keep the biological characteristics of glycosylation, disulfide bond and the like.
Another neurotrophic factor, brain cell-derived neurotrophic factor (BDNF, the same below), is a functional neurotrophic factor produced and secreted by brain cells. BDNF has wide neurotrophic effect on a plurality of nerve cells in the center and the periphery, such as promoting the survival and differentiation of certain nerve groups, regulating and controlling the bremsstrahlung force of neurite axis, relating to the learning, memory and spinal cord pain mechanism of hippocampus, and the like. More importantly, BDNF plays an important role in the survival and growth of dopaminergic neurons. It has effects in preventing apoptosis cell death, increasing survival of neuron positive to tyrosine hydrolase and growth of neuron fiber. BDNF is therefore considered as a good candidate for the treatment of certain central nervous system disorders as a potent neuroprotective factor.
Since the expression of the two neurotrophic factors in vivo is less or absent, it is an urgent need in the field of medicine to obtain a large amount of neurotrophic factors with high activity by in vitro production. At present, the realization of in vitro production of neurotrophic factors by recombinant expression has been reported, for example, prokaryotic expression of neurotrophic factors by escherichia coli is a common method (see CN 107793485, CN 101775072A and CN 101775072A), however, the production of neurotrophic factors by prokaryotic expression requires inducible expression, and after expression of inclusion bodies, multi-step purification, which is complicated to operate, and has low expression level and low activity. At present, there are reports of recombinant expression of neurotrophic factors by eukaryotic expression systems, for example, chinese patent publication No. CN 1364812a, entitled human glial cell-derived neurotrophic factor and derivatives thereof, and applications thereof, report that transfection of target genes into CHO cells by expression vector pSVT7, screening of GDNF-expressing cell lines, and purification by heparin affinity chromatography, ion exchange chromatography, etc. not only are the operation complicated, but also the yield is low.
Disclosure of Invention
In view of the above, the present invention aims to provide an expression recombinant neurotrophic factor fusion protein and a preparation method thereof, which have the characteristics of stable and high expression and are simple and convenient in separation and purification operations.
The invention also aims to provide the recombinant neurotrophic factor fusion protein which is not only close to the dimer structure of the natural protein, but also has higher biological activity and provides a high-quality material for the subsequent preparation of related medicaments.
The invention provides a fusion gene of a recombinant neurotrophic factor, which comprises a neurotrophic factor gene sequence, a secretory signal peptide TPA gene sequence and an Fc gene sequence.
Preferably, the fusion gene of the recombinant neurotrophic factor is a secretion signal peptide TPA gene sequence-Fc gene sequence-neurotrophic factor gene sequence or a secretion signal peptide TPA gene sequence-neurotrophic factor gene sequence-Fc gene sequence.
Preferably, the neurotrophic factor is GDNF or BDNF;
the nucleotide sequence of the GDNF is shown as SEQ ID NO. 6, and the nucleotide sequence of the BDNF is shown as SEQ ID NO. 8.
Preferably, the secretory signal peptide TPA gene sequence is shown as SEQ ID NO. 9, and the Fc gene sequence is shown as SEQ ID NO. 10.
Preferably, the fusion gene of the recombinant GDNF is TPA gene sequence-Fc gene sequence-GDNF gene sequence, and the nucleotide sequence is shown as SEQ ID NO: 11;
the fusion gene of the recombinant BDNF is a TPA gene sequence-BDNF gene sequence-Fc gene sequence, and the nucleotide sequence is shown in SEQ ID NO. 12.
The invention provides a recombinant expression vector comprising the fusion gene.
Preferably, the backbone vector is pPBml-PNCE.
The invention provides a recombinant cell comprising the fusion gene or the recombinant expression vector.
Preferably, the recombinant cell is a HEK293 cell.
The invention provides a method for preparing the recombinant neurotrophic factor fusion protein, which comprises the following steps:
1) constructing a recombinant expression vector containing the fusion gene;
2) transforming the recombinant expression vector in the step 1) into a mammalian cell, screening a high-expression cell strain from the obtained recombinant cell, culturing, separating supernatant, and purifying to obtain the recombinant expression neurotrophic factor fusion protein.
Preferably, the method for screening the high-expression cell strain comprises the steps of detecting the expression quantity of the fusion protein by using an Anti-Fc Elisa detection kit, and selecting the cell strain with the highest protein expression quantity for culture.
Preferably, the purification is performed by using a ProteinA column, and the Loading buffer containing Igepal CA-630 is used for removing endotoxin in the protein.
Preferably, the mass concentration of the Igepal CA-630 is 0.1-2%.
Preferably, the flow rate of the Loadingbuffer containing the Igepal CA-630 is 0.4-2 mL/min.
The invention provides a fusion protein coded by a fusion gene of the recombinant neurotrophic factor, wherein the fusion protein is a dimer; the fusion protein includes a neurotrophic factor and an Fc structure.
The invention provides application of the recombinant expression neurotrophic factor fusion protein in promoting the growth and/or differentiation of neuronal cells.
Preferably, the neurons comprise dopaminergic neurons and/or motor neurons.
The invention provides a method for recombining and expressing a neurotrophic factor, which takes a plasmid containing a high-expression promoter enhancer as a carrier skeleton, constructs a high-expression recombination carrier by a fusion gene formed by the neurotrophic factor, a secretion signal peptide TPA and Fc, and can improve the extracellular secretion efficiency of a target protein and the yield of the target protein; the constructed recombinant vector is transformed into a mammalian cell expression system, and because the vector carries a secretion signal peptide TPA, a target protein can be secreted out of cells, and the culture supernatant can be directly harvested for purification, so that the expression step is simplified and the purity of the obtained protein is higher; the obtained target protein is closer to a natural product in function than a prokaryotic expression product; meanwhile, the Fc structure of the invention is expressed together with the target protein, and the obtained recombinant target protein not only can form a natural dimer structure, but also has better biological activity, and can support the growth and differentiation of various cells such as motor neurons, dopaminergic neurons and the like and the application of related diseases on medicaments. In the experiment, the yield is generally calculated on the nanogram level, and according to the method provided by the invention, 1-2 mg of GDNF/BDNF with high biological activity and high purity (more than 95%) can be prepared by purifying every 100mL of culture supernatant, so that the GDNF/BDNF prepared by the method can greatly meet the market demand.
Meanwhile, the method provided by the invention improves the yield of the target protein and reduces the production cost under the condition of a certain usage amount of raw materials; the recombinant protein prepared by the invention is secreted, exists in a cell culture solution, is purified subsequently, a plurality of affinity chromatographic columns are not needed, and the target protein with higher purity (more than 95 percent) can be obtained by one-step purification, so that the time cost and the economic cost on a purification process are saved while the operation is simplified;
further, the present invention specifically defines that the neurotrophic factor comprises GDNF or BDNF. Experiments prove that the insertion position of Fc has obvious influence on the protein expression quantity and purity, namely the cell strain protein expression quantity and purity for constructing TPA-Fc-GDNF and TPA-BDNF-Fc sequences are higher, and the clinical-grade use and purification requirements can be met; the experimental result also shows that the purity of the recombinant protein prepared by constructing the plasmids of TPA-GDNF-Fc and TPA-Fc-BDNF is less than 50 percent, and the expression amount is relatively low.
Further, the method provided by the invention specifically defines the purification process. The current preparation process for recombinant neurotrophin fusion proteins involves only expression and purification steps, without removal of endotoxins. The endotoxin content is an important quality attribute of the recombinant protein and is also a key index for whether the recombinant protein can be applied to the production of clinical-grade products. The recombinant protein has high endotoxin content, toxic effect on cells and poor cell growth, and cannot be applied to scientific research and clinic. In the purification process, the Igepal CA-630 is used for removing endotoxin, so that the endotoxin content (0.1 EU/mu g) of the target protein is effectively reduced, and the study and the application in the aspects of nerve growth, development and the like are facilitated; and the operation is simple, the amplification is convenient, and the production requirement can be met.
Drawings
FIG. 1 is a plasmid map of high expression GDNF constructed in the present invention; wherein, FIG. 1-1 is a plasmid map of pPBml-PNC-TPA-Fc-GDNF, and FIG. 1-2 is a plasmid map of pPBml-PNC-TPA-GDNF-Fc;
FIG. 2 is a plasmid map of high expression BDNF constructed by the invention; wherein, figure 2-1 is the plasmid map of plasmid pPBml-PNC-TPA-BDNF-Fc, figure 2-2 is the plasmid map of plasmid pPBml-PNC-TPA-Fc-BDNF;
FIG. 3 shows the cell growth status after screening the monoclone and expansion of the high expression cell strain according to the present invention;
FIG. 4 is a SDS-PAGE analysis of GDNF secreted by positive cell lines constructed from pPBml-PNC-TPA-Fc-GDNF according to the present invention (reduced and non-reduced represent eluted samples treated with and without reducing agent, respectively);
FIG. 5 is a SDS-PAGE analysis of GDNF secreted by positive cell lines constructed from pPBml-PNC-TPA-GDNF-Fc of the present invention (reduced and non-reduced represent eluted samples treated with and without reducing agent, respectively);
FIG. 6 is a Westernblot analysis of GDNF obtained in accordance with the present invention;
FIG. 7 is a SDS-PAGE analysis of BDNF obtained with pPBml-PNC-TPA-Fc-BDNF and pPBml-PNC-TPA-BDNF-Fc of the present invention (reduced and non-reduced representing eluted samples treated with and without reducing agent, respectively), wherein the left panel is a SDS-PAGE analysis of BDNF expressed by pPBml-PNC-TPA-Fc-BDNF and the right panel is a SDS-PAGE analysis of BDNF expressed by pPBml-PNC-TPA-BDNF-Fc;
FIG. 8 shows the Westernblot analysis results of BDNF obtained by the present invention;
FIG. 9 is a graph of the detection of biologically active EC by C6 cells for GDNF obtained in the present invention50Results of (A), (B)Stemcell and R&D is the detection result of commercial GDNF respectively);
FIG. 10 shows that the BDNF obtained by the invention uses C6 cells to detect the biological activity EC50The result of (1);
FIG. 11 shows the commercial BDNF biological Activity EC from Abcam corporation50The detection result of (3);
FIG. 12 shows that GDNF or BDNF obtained by the present invention has no adverse effect on the maturation of motor neurons and dopaminergic neurons, wherein FIG. 12A shows the status of mature motor neurons and the expression of specific markers cultured using self-produced and R & D GDNF or BDNF, and FIG. 12B shows the status of mature dopaminergic neurons and the expression of specific markers cultured using self-produced and R & D GDNF or BDNF.
Detailed Description
The invention provides a fusion gene of a recombinant neurotrophic factor, which comprises a neurotrophic factor gene sequence, a secretory signal peptide TPA gene sequence and an Fc gene sequence.
In the present invention, the fusion gene of the recombinant neurotrophic factor is preferably a secretion signal peptide TPA gene sequence-Fc gene sequence-neurotrophic factor gene sequence or a secretion signal peptide TPA gene sequence-neurotrophic factor gene sequence-Fc gene sequence. The amino acid sequence of the secretory signal peptide TPA is shown as SEQ ID NO. 3
(MDAMKRGLCCVLLLCGAVFVSPS) and the nucleotide sequence is shown as SEQ ID NO:9 (ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGC). Compared with the fusion protein without TPA, the addition of the secretory signal peptide TPA obviously improves the expression level of GDNF or BDNF. The amino acid sequence of the Fc is shown as SEQ ID NO. 4(PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), and the nucleotide sequence is shown as SEQ ID NO. 10 (CCAAAGAGCTGCGACAAGACACACACCTGCCCACCATGTCCAGCACCTGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCTCCAAAGCCCAAGGATACACTGATGATCTCTAGGACCCCTGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCAGAGGTGAAGTTTAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTATCGCGTGGTGTCTGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGTAAGGTGTCCAATAAGGCCCTGCCAGCCCCCATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCAAGGGAGCCTCAGGTGTACACACTGCCCCCTTCCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCCCGGCAAG). The insertion of the Fc structure significantly improves the formation efficiency of GDNF or BDNF dimer, simplifies the purification steps, improves the protein purity and has better biological activity.
In the present invention, the neurotrophic factor preferably comprises GDNF or BDNF. The amino acid sequence of GDNF is shown as SEQ ID NO:5(MDAMKRGLCCVLLLCGAVFVSPSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKTRMSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI), and the nucleotide sequence is shown as SEQ ID NO:6 (ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCCAAAGAGCTGCGACAAGACACACACCTGCCCACCATGTCCAGCACCTGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCTCCAAAGCCCAAGGATACACTGATGATCTCTAGGACCCCTGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCAGAGGTGAAGTTTAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTATCGCGTGGTGTCTGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGTAAGGTGTCCAATAAGGCCCTGCCAGCCCCCATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCAAGGGAGCCTCAGGTGTACACACTGCCCCCTTCCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCCCGGCAAGACGCGTATGAGCCCCGATAAGCAGATGGCCGTGCTGCCTCGGAGAGAGAGGAACAGGCAGGCAGCAGCAGCAAACCCAGAGAATTCCAGGGGCAAGGGCAGGCGCGGACAGAGGGGCAAGAACAGAGGCTGCGTGCTGACCGCCATCCACCTGAATGTGACAGATCTGGGCCTGGGCTACGAGACCAAGGAGGAGCTGATCTTCCGGTATTGCAGCGGCTCCTGTGATGCCGCCGAGACCACATACGACAAGATCCTGAAGAACCTGTCTCGGAATCGGAGACTGGTGAGCGACAAAGTGGGCCAGGCCTGCTGTAGACCCATCGCCTTCGACGATGACCTGTCCTTTCTGGATGACAATCTGGTGTATCACATCCTGAGGAAGCACTCTGCCAAGCGGTGCGGCTGTATCTGA). The amino acid sequence of BDNF is shown as SEQ ID NO. 7(MHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGRTRPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK), and the nucleotide sequence is shown as SEQ ID NO. 8 (ATGGATGCAATGAAGAGAGGGCTCTGCTGTGTGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCATGCACTCCGACCCAGCAAGGAGAGGAGAGCTGAGCGTGTGCGATAGCATCTCCGAGTGGGTGACCGCCGCCGACAAGAAGACAGCCGTGGATATGAGCGGCGGCACCGTGACAGTGCTGGAGAAGGTGCCCGTGTCCAAGGGCCAGCTGAAGCAGTACTTCTATGAGACCAAGTGCAACCCTATGGGCTACACAAAGGAGGGCTGTAGGGGCATCGACAAGCGCCACTGGAATTCTCAGTGTCGGACCACACAGAGCTATGTGCGGGCCCTGACCATGGACTCTAAGAAGAGAATCGGCTGGCGGTTTATCAGAATCGATACATCCTGCGTGTGCACCCTGACAATCAAGAGGGGCCGCACGCGTCCAAAGAGCTGCGACAAGACACACACCTGCCCACCATGTCCAGCACCTGAGCTGCTGGGAGGACCAAGCGTGTTCCTGTTTCCTCCAAAGCCCAAGGATACACTGATGATCTCTAGGACCCCTGAGGTGACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCAGAGGTGAAGTTTAACTGGTACGTGGACGGCGTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACTCCACCTATCGCGTGGTGTCTGTGCTGACAGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTATAAGTGTAAGGTGTCCAATAAGGCCCTGCCAGCCCCCATCGAGAAGACCATCTCTAAGGCAAAGGGACAGCCAAGGGAGCCTCAGGTGTACACACTGCCCCCTTCCAGAGACGAGCTGACCAAGAACCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCCGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCCGATGGCTCTTTCTTTCTGTATTCCAAGCTGACCGTGGACAAGTCTCGGTGGCAGCAGGGCAACGTGTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTACACACAGAAGTCTCTGAGCCTGTCCCCCGGCAAGTGA). The order of GDNF fusion genes is preferably TPA gene sequence-Fc-GDNF gene sequence or TPA gene sequence-GDNF gene sequence-Fc gene sequence, more preferably TPA gene sequence-Fc gene sequence-GDNF gene sequence. The BDNF fusion gene sequence is preferably TPA gene sequence-BDNF gene sequence-Fc gene sequence or TPA gene sequence-Fc gene sequence-BDNF gene sequence, more preferably TPA gene sequence-BDNF gene sequence-Fc. The nucleotide sequence of the TPA gene sequence-Fc gene sequence-GDNF gene sequence is shown in SEQ ID NO: 11. The nucleotide sequence of the TPA gene sequence-BDNF gene sequence-Fc gene sequence is shown in SEQ ID NO. 12.
The invention provides a recombinant expression vector comprising the fusion gene. The backbone vector is preferably pPBml-PNCE.
The invention provides a recombinant cell comprising the fusion gene or the recombinant expression vector. The recombinant cell is preferably a HEK293 cell.
The invention provides a fusion protein coded by a fusion gene of the recombinant neurotrophic factor, wherein the fusion protein is a dimer; the fusion protein includes a neurotrophic factor and an Fc structure. The fusion protein obtained by cell expression of the fusion gene vector shown by TPA-Fc-GDNF or TPA-GDNF-Fc is GDNF dimer, wherein the protein expression amount and purity of the cell expression of the TPA-Fc-GDNF-containing vector are higher. The amino acid sequence of the fusion protein GDNF (Fc-GDNF) is shown in SEQ ID NO: 1. The fusion protein obtained by cell expression of the fusion gene vector shown by TPA-Fc-BDNF or TPA-BDNF-Fc is BDNF dimer, wherein the protein expression amount and purity of the TPA-BDNF-Fc-containing vector expressed by cells are higher. The amino acid sequence of the fusion protein BDNF (BDNF-Fc) is shown in SEQ ID NO. 2.
The invention provides a method for expressing the recombinant neurotrophic factor fusion protein, which comprises the following steps:
1) constructing a recombinant expression vector containing the fusion gene;
2) transforming the recombinant expression vector in the step 1) into mammalian cells, screening high-expression cell strains from the obtained recombinant cells, culturing, separating supernatant, and purifying to obtain the recombinant expression neurotrophic factor.
The invention constructs a recombinant plasmid containing the fusion gene.
In the present invention, the backbone vector of the recombinant plasmid is preferably pPBml-PNCE. The source of the pPBml-PNCE is not particularly limited in the present invention, and a source of pPBml-PNCE known in the art may be used. In the present examples, the pPBml-PNCE was the same plasmid vector as the pPBml-CCP plasmid described in the CN111500629A patent. Experiments prove that different protein expression forms require different expression vectors, and the proteins obtained by the same protein sequence on different expression vectors are not completely the same. Compared with the conventional vectors (pGEX-2T, pET-45b, pSVT-7, pET-28b, pET-32a and pPBml-PNEE), the pPBml-PNCE is used as a framework vector, and the expression quantity of the GDNF and BDNF fusion protein is obviously improved.
The method for constructing the recombinant vector is not particularly limited in the present invention, and the method for constructing the recombinant vector known in the art, such as enzyme digestion, ligation, and verification, may be used. In the present example, when pPBml-PNCE is a backbone vector, Xba I and BamHI are preferable as the multiple cloning sites into which the fusion protein sequence is inserted. The ligation is preferably performed using T4 ligase. Preferably, the verification method comprises the steps of transforming the ligation product into escherichia coli, screening and culturing, extracting plasmids after selecting positive transformants, and performing double enzyme digestion verification and sequencing. In the embodiment of the invention, in the constructed recombinant vectors, pPBml-PNC-TPA-Fc-GDNF, pPBml-PNC-TPA-BDNF-Fc and pPBml-PNC-TPA-Fc-BDNF can successfully express the dimer target protein with high biological activity, but pPBml-PNC-TPA-GDNF and pPBml-PNC-TPA-BDNF construct plasmids have extremely small amounts of GDNF or BDNF dimer in cell expression. Therefore, the pPBml-PNCE vector and the fusion gene formed by the neurotrophic factor, TPA and Fc in HEK293 cells can ensure correct expression and obtain highly biologically active proteins.
After obtaining the recombinant plasmid, the recombinant plasmid is transformed into mammalian cells, high-expression cell strains are screened and cultured, supernatant is separated, and the recombinant expression neurotrophic factor is obtained through purification.
The method of the transformation is not particularly limited in the present invention, and a transformation method well known in the art, such as electric transformation, may be used. After transformation, screening for positive clones is preferred. The screening method is preferably carried out using G418. The method for screening the high-expression cell strain preferably comprises the steps of detecting the expression quantity of the fusion protein by using an Anti-Fc Elisa detection kit, and selecting a cell strain with the highest protein expression quantity for culture. The method of separating the supernatant is preferably centrifugation. The centrifugation conditions are preferably 200g, and the centrifugation time is 5 min. After collecting the supernatant, the protein was washed. The washed protein was purified.
In the present invention, the purification is preferably performed by using a ProteinA column, and endotoxin in the protein is removed by using a Loadingbuffer containing Igepal CA-630. The concentration of the Igepal CA-630 is preferably 0.1-2%, more preferably 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5%, 2%, and most preferably 1%. The flow rate for removing endotoxin may be 0.4mL/min, 0.6mL/min, 0.8mL/min, 1mL/min, 1.2mL/min, 1.5mL/min, 2mL/min, but is not limited thereto, and the flow rate is 0.4 to 2mL/min, more preferably 1 mL/min. Compared with other reagents (C7BzO, Triton X114, isopropanol, 1, 2-hexanediol, ASB-14, CHAPS), Igepal CA-630 had the best endotoxin removal effect.
The recombinant fusion protein GDNF/BDNF, the recombinant GNDF/BDNF protein and the dimeric GDNF/dimeric BDNF are all indicated to be the recombinant neurotrophic factor fusion protein (recombinant GDNF/BDNF fusion protein) prepared by the method.
In the present invention, for example, "GDNF/BDNF" means GDNF or BDNF, and can be used for the expression on genes or proteins.
In view of the fact that the prepared recombinant expression neurotrophic factor has higher biological activity, the invention provides the application of the recombinant expression neurotrophic factor in promoting the growth and/or differentiation of neuronal cells.
In the present invention, the neurons preferably comprise dopamine neurons and/or motor neurons.
The following examples are provided to illustrate the method of expressing the recombinant neurotrophin fusion protein, and the use thereof, but they should not be construed as limiting the scope of the present invention.
The buffer reagent composition during protein purification is shown in table 1:
TABLE 1 buffer reagent Components
Serial number Composition (I) Working concentration
1 Glycerol (Glycerol) 10～60％
2 MgSO 4 1～2mM
3 K2HPO4 50～72mM
4 KH2PO4 10～17mM
5 Ampicillin 20～100μg/mL
6 Protein loading buffer 6ⅹ
7 Acrylamide 10～30％
8 SDS 8～12％
9 Ammonium persulfate 5～10％
10 TEMED 0.04～0.1％
12 Protein marker 12～180KDa
13 Tris-Hcl 5～200mM
14 NaCl 20～500mM
15 IgepalCA-630 0.1～5％
16 Glycine 20～500mM
17 ProteinA 1～5mL
18 G418 100～1000μg/mL
19 HCl 15～30％
20 NaOH 0.1～0.5M
21 Ethanol 20％
22 Sodium acetate 10～50mM
TABLE 2 Loading buffer reagent Components
Serial number Composition (I) Working concentration
1 Tris-Hcl 5～200mM
2 NaCl 20～500mM
3 HCl 15～30％
TABLE 3 Elute buffer reagent compositions
Serial number Composition (I) Working concentration
1 Glycine 20～500mM
2 NaCl 20～500mM
TABLE 4 Neutralize buffer reagent compositions
Serial number Composition (I) Working concentration
1 Tris-Hcl 5～200mM
2 HCl 15～30％
Example 1
Construction method of high expression plasmid pPBml-PNC-TPA-Fc-GDNF and pPBml-PNC-TPA-BDNF-Fc
1.1 plasmid cleavage
The existing pPBml-PNCE plasmid and synthetic TPA-Fc-GDNF or TPA-BDNF-Fc fusion protein sequence (synthesized by Nanjing Kinshire) were digested at 37 ℃ for 2h as shown in the following tables 5 and 6, wherein the endonuclease comprises Xba I (manufacturer: NEB, cat # R0145L) and BamHI (manufacturer: NEB, cat # R3131L), and Cutsmart is a component of the endonuclease kit.
TABLE 5 pPBml-PNCE plasmid cleavage System
Components Volume of
XbaI 3μL
BamHI 3μL
pPBml-PNCE 10μL
Cutsmart 3μL
ddH2O 11μL
TABLE 6 fusion protein sequence cleavage System
Components Volume of
XbaI 5μL
BamHI 5μL
TPA-Fc-GDNF or TPA-BDNF-Fc 30μL
Cutsmart 5μL
ddH2O 5μL
1.2DNA fragment recovery and ligation
The DNA was recovered by using a DNA purification recovery kit, and the details were referred to the kit manual (manufacturer: Tiangen organism, cat # DP 209-03). After the DNA was recovered, ligation was performed (T4 DNA ligase, manufacturer: NEB, cat # M0202V), and the ligation system is shown in Table 7. The ligation procedure was 22 ℃ ligation for 60 min.
TABLE 7 connection system
Components Volume of
pPBml-PNC recovery fragment 3μL
TPA-Fc-GDNF recovery fragments or TPA-BDNF-Fc recovery fragments 14μL
T4DNA ligase 1μL
T4DNA ligase buffer (10 ×) 2μL
1.3 ligation product transformation and plasmid validation
Transformation system: adding the ligation product into Trans5 alpha competent cells (manufacturer: Beijing holotype gold, cat # CD201-02), performing ice bath for 30min, performing heat shock at 42 ℃ for 90s, performing ice bath for 3min, adding 500uL LB culture medium, and performing shake culture at 37 ℃ and 250rpm for 30 min; 12000rpm/min, centrifugation for 1min, leaving 100 μ L of supernatant, resuspending the thallus, coating on an ampicillin resistant LB solid plate, and culturing in a biochemical incubator at 37 ℃ for 16-18 h. The monoclonal antibody is picked the next day, cultured in a shaking incubator at 37 ℃ and 250rpm for 10h, and the plasmid is extracted by a small plasmid extraction kit, wherein the specific plasmid extraction step refers to the kit instruction (manufacturer: Tiangen organism, cargo number: DP 103-03). Xba I and BamHI were verified by double digestion and the plasmid was sequenced. Thus, the successfully constructed pPBml-PNC-TPA-Fc-GDNF (figure 1-1) or pPBml-PNC-TPA-BDNF-Fc plasmid (figure 2-1) is obtained.
Example 2
Construction and screening of high expression cell line
1. Electrotransfer and positive clone screening
1.1 plasmid electrotransformation
Selecting HEK293 cells (purchased from Kjeri Biotech Co., Ltd.) with a survival rate of more than 95%, and selecting 1 × 106The cells were centrifuged to remove the supernatant to obtain cells. Resuspending cells with 100. mu.L electrotransfer Buffer 10, adding 1. mu.g of pPBml-PNC-TPA-Fc-GDNF or pPBml-PNC-TPA-BDNF-Fc and pEhypbase plasmid, mixing, adding blank cell control group (without plasmid), transferring into an electrotransfer cup, electrotransfering with Lonza electrotransfer instrument (adjusted to HEK293 ATCC mode), resuspending cells with DMEM + 10% FBS culture medium after electrotransfer, spreading in 6-well plate, placing at 37 ℃, and 5% CO2Culturing in an incubator with concentration and saturation humidity.
1.2 Positive clone screening
After 24h adherent growth, adherent HEK293 cells were digested with Solase cell digest, resuspended in DMEM + 10% FBS, cell density was adjusted after manual counting, and 800 μ G/mL G418 was added and plated in 24-well plates at 2 × 104cells/well, 1 mL/well, equal volume change every two days. G418 was killed for 10 days, and positive clones were observed. G418 is removed, DMEM + 10% FBS culture medium is added for continuous culture, 3-5 cells (remarks: GDNF positive clone cells or BDNF positive clone cells) are frozen and stored for subsequent tests when the growth of the cells is more than 50%.
Example 3
Construction method of recombinant plasmid pPBml-PNC-TPA-GDNF-Fc (spectrogram shown in figure 1-2) and pPBml-PNC-TPA-Fc-BDNF (spectrogram shown in figure 2-2)
The pPBml-PNCE plasmid and the synthetic TPA-GDNF-Fc or TPA-Fc-BDNF fusion protein sequence were used to construct recombinant vectors according to the method of example 1. Positive clones were obtained according to the method of example 2.
Example 4
Screening of high expression cell lines
G418 screening the positive cell population obtained in examples 2 and 3, until they grow over 50%, digesting into single cells with Solase cell digest, resuspending with DMEM + 10% FBS medium, counting Vi-cells, diluting to the number of cells of the lowest growth condition by limiting dilution method, inoculating 100. mu.L/well into 96-well plate, placing at 37 deg.C, and 5% CO2Culturing in an incubator with concentration and saturation humidity. And culturing for 12 days, selecting a monoclonal antibody with the confluency of more than 50%, transferring the monoclonal antibody to a 24-well plate, and performing amplification culture by using a DMEM + 10% FBS culture medium.
The growth of the monoclonals and the morphology of the cells in suspension culture after transfer of the monoclonals into 6-well plates for expansion are shown in FIG. 3.
After 5 days in 24-well plates, the plates were expanded to 6-well plates, and the expansion was continued with a serum-free KOP293 medium (purchased from Kyoto Biotech Co., Ltd.). Selecting cell strain with good cell density of 80%, counting Vi-cell, and adjusting cell density to 1 × 106cells/mL, 4 mL/well, inoculating to 6-well plate for suspension culture for 5 days, collecting 0.5mL cell supernatant respectively, and detecting protein expression condition by Anti-Fc Elisa detection kit. According to the detection result, a cell with the highest expression amount of GDNF or BDNF is screened from positive cells constructed by pPBmL-PNC-TPA-Fc-GDNF, pPBmL-PNC-TPA-GDNF-Fc, pPBml-PNC-TPA-GDNF and pPBml-PNC-TPA-Fc-BDNF respectively, and the culture and the cryopreservation are continued to be expanded. The ELISA assay results are shown in Table 8.
TABLE 8 ELISA test results
Figure BDA0003360027800000101
Figure BDA0003360027800000111
In Table 8, different values represent ELISA assay levels, and higher absorbance values indicate higher expression of the protein of interest. Wherein all clones of TPA-GDNF-Fc and TPA-Fc-BDNF are low in expression; the expression of the TPA-Fc- GDNF clones 1,2, 5 and 7 is high, wherein the TPA-Fc-GDNF clone 1 is a pPBmL-PNC-TPA-Fc-GDNF high expression cell strain obtained by screening; the expression of the TPA-BDNF- Fc clone 1, 3 and 6 is high, wherein the TPA-BDNF-Fc clone 6 is a pPBmL-PNC-TPA-BDNF-Fc high expression cell strain obtained by screening.
Example 5
The high expression cell line selected in example 4 was inoculated and the recombinant protein was isolated and purified
1. Crude protein separation
Selecting TPA-Fc-GDNF clone 1 and TPA-BDNF-Fc clone 6 as GDNF or BDNF high expression cell strains until the cell density reaches 6-8 multiplied by 106cells/mL, operating as follows: inoculation density 1X 106cells/mL, 250mLKOP293 medium, 1L shake flask, horizontal shaker rotation 120 rpm. Standing at 37 deg.C for 5% CO2Culturing in an incubator with concentration and saturation humidity.
Culturing for 4-5 days until the cell density reaches 6-8 multiplied by 106cells/mL stopped culturing, and the supernatant was collected by centrifugation at 200g for 5 min. The collected supernatant was aliquoted into dialysis bags (the dialysis bags were previously boiled in water for 10min), each dialysis bag was about 50mL, and the bags were dialyzed with a 5-fold volume Loadingbuffer by a magnetic stirrer at 400rpm for 2 hours at room temperature. After dialysis, the supernatant was collected by centrifugation at 12000g for 10min at room temperature and used for affinity chromatography. If the culture supernatant obtained by collection has a large volume, an ultrafiltration concentration device can be considered, and the specific operation is as follows: the cell density reaches 6-8 x106Stopping culturing cells/mL, centrifuging at 200g for 5min, and collecting supernatant; concentrating the collected supernatant by 20 times by using an ultrafiltration concentration device, then adding 20 times of Loadingbuffer in volume, uniformly mixing, concentrating by using the ultrafiltration concentration device by 20 times, and directly using the concentrated product for affinity chromatography.
2. Protein purification
The ProteinA column was pre-equilibrated with Loadingbuffer (100mM Tris-HCl, 150mM NaCl, pH 7.5), approximately 30mL, 2 mL/min. The treated cell supernatant was slowly added to the ProteinA column, and the flow rate was controlled at 0.4 mL/min. After the sample loading is finished, adding Loadingbuffer to remove the hybrid protein until OD280<0.02 mg/mL; adding Loadingbuffer containing Igepal CA-630 to remove endotoxin, wherein the volume is about 60mL, and the flow rate is 1 mL/min; add Loadingbuffer to remove Igepal CA-630 until OD280<Up to 0.02 mg/mL. After washing, the GDNF/BDNF protein was eluted thoroughly by adding the protein eluent, elubeffer (100mM Glycine, 150mM nacl, pH 3), and the protein solution was collected in 1.5mL centrifuge tubes, 1 mL/tube, and left overnight in a 4-degree refrigerator. After 24h Neutralizebuffer (100mM Tris-HCl, pH 8.0) was added at a ratio of 1:1 to neutralize the pH of the protein. After elution GDNF or BDNF was obtained for SDS-PAGE analysis.
The NaCl concentration of the Loadingbuffer is too high, so that the target protein is not combined with the ProteinA column, and the impurities such as impurity protein, micromolecule and the like are not completely removed due to too low concentration, so that the purity of the target protein is influenced. The NaCl concentration in the Loadingbuffer may be 20mM, 50mM, 100mM, 150mM, 200mM, 300mM, or 500mM in other embodiments, but is not limited thereto, and the NaCl concentration is 20 to 500mM, preferably 150 mM. After dialysis, the supernatant was collected by centrifugation for 10 min.
The flow rate for loading the protein may be 0.2mL/min, 0.4mL/min, 0.6mL/min, 0.8mL/min, 1mL/min, 2mL/min, but is not limited thereto, and the flow rate is 0.2-2 mL/min, preferably 0.4 mL/min. And after the loading is finished, adding Loadingbuffer to remove the hybrid protein.
The endotoxin is removed on the column, and the Loadingbuffer containing the Igepal CA-630 is added to remove the endotoxin, wherein the concentration of the Igepal CA-630 can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.2%, 1.5% and 2%, but the concentration of the CA-630 is 0.1-2%, and 1% is preferred in the invention. The flow rate for removing endotoxin may be 0.4mL/min, 0.6mL/min, 0.8mL/min, 1mL/min, 1.2mL/min, 1.5mL/min, 2mL/min, but is not limited thereto, and the flow rate is 0.4-2 mL/min, preferably 1 mL/min.
The SDS-PAGE electrophoresis results of recombinant GDNF secreted by positive cell lines constructed from pPBmL-PNC-TPA-Fc-GDNF are shown in FIG. 4. As can be seen from FIG. 4, the reduced SDS was present in the form of a monomer; non-reducing SDS 95% was present as dimer, no protein in monomeric form, only a small amount of protein formed a high polymer (< 5%). Indicating that 95% of the purified protein was present as an active dimer. By concentration determination, 1-2 mg GDNF with high biological activity can be prepared by purifying every 100mL culture supernatant. The SDS-PAGE electrophoresis results of the recombinant GDNF secreted by the positive cell strain constructed by pPBml-PNC-TPA-GDNF-Fc are shown in FIG. 5. The GDNF expressed by the expression plasmid pPBml-PNC-TPA-GDNF-Fc not only has little amount of harvested dimer, but also has lower purity. GDNF was purified to less than 0.1mg per 100mL of culture supernatant as determined by concentration. Western Blot identification of recombinant GDNF secreted by positive cell lines constructed from pPBmL-PNC-TPA-Fc-GDNF is shown in FIG. 6.
The SDS-PAGE electrophoresis result of the recombinant BDNF secreted by the positive cell strain constructed by pPBmL-PNC-TPA-BDNF-Fc and the SDS-PAGE electrophoresis result of the recombinant GDNF secreted by the positive cell strain constructed by pPBmL-PNC-TPA-Fc-BDNF are shown in figure 7. The recombinant BDNF secreted by the positive cell strain constructed by pPBmL-PNC-TPA-BDNF-Fc in each 100mL of culture supernatant is 1-2 mg, and the recombinant BDNF secreted by the positive cell strain constructed by pPBmL-PNC-TPA-Fc-BDNF is lower than 0.1 mg. The expression level of the recombinant BDNF secreted by the positive cell strain constructed by the pPBmL-PNC-TPA-BDNF-Fc is obviously higher than that of the recombinant protein of the positive cell strain constructed by the pPBmL-PNC-TPA-Fc-BDNF. The results of the WesternBlot identification of recombinant BDNF are shown in figure 8.
Example 6
Protein bioactivity assay
Selecting well-grown C6 cells, digesting with Solase cell digest solution, counting Vi-cells, adjusting cell density to 5x103cells/mL were seeded at 500 cells/well, 100. mu.L/well in 96-well plates. GDNF/BDNF protein obtained from the production of example 6 was added to each well individually, and final protein concentrations of 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 200ng/mL were set, with 3 replicates per group. Standing at 37 deg.C for 5% CO2Culturing for 72h in an incubator with the concentration and saturation humidity. 10 μ L/well CCK-8Solution, mixing by gentle shaking, standing at 37 deg.C with 5% CO2Incubating for 1h in an incubator with concentration and saturation humidity, detecting OD450 light absorption value by an enzyme labeling instrument, and calculating GDNF or BDNF protein EC by an Excel line graph50The value is obtained.
The results of the biological activity assay of the recombinant GDNF protein secreted by the positive cell strain constructed by pPBmL-PNC-TPA-Fc-GDNF are shown in FIG. 9. The results show that GDNF EC of the present invention50The value is 0.01-0.02. mu.g/mL, the recombinant GDNF protein (cat # 78058) from Stemcell is purchased from Escherichia coli prokaryotic expression system, and the EC thereof50Value of 3. mu.g/mL, GDNF EC of the present invention50The value is significantly less than GDNF of Stemcell company, which shows that the biological activity of GDNF is significantly higher than that of GDNF of Stemcell company; purchase R&Recombinant GDNF protein (cat # 212-GD) from company D, derived from mouse myeloma cell expression system, its EC50The value is about 0.01 mu g/mL, the difference is not obvious from the invention, 1-2 mg GDNF protein can be purified by 100mL of the invention, considering that R&The price of GDNF from D company, the present invention is still more cost effective.
The result of the biological activity detection of the recombinant BDNF protein secreted by the positive cell strain constructed by pPBmL-PNC-TPA-BDNF-Fc is shown in figure 10, the recombinant BDNF protein (cargo number: ab206642) purchased from Abcam company is derived from an escherichia coli prokaryotic expression system, and the result of the biological activity detection is shown in figure 11. The results show that the BDNF EC of the invention50A value of 0.01 to 0.02. mu.g/mL, a recombinant BDNF EC of Abcam50The value is 0.5-0.8 mu g/mL, the BDNF EC of the invention50The value is obviously smaller than that of recombinant BDNF of an Abcam company, which shows that the biological activity of the BDNF is obviously higher than that of the recombinant BDNF of the Abcam company; meanwhile, 1-2 mg of BDNF protein can be purified by 100mL of the invention, and the invention has more obvious advantage than the above method in cost in view of the price of the recombinant BDNF of Abcam company.
The biological activity detection results of GDNF protein and BDNF protein secreted by positive cell strains constructed by pPBml-PNC-TPA-GDNF and pPBml-PNC-TPA-Fc-BDNF show that: the activity of GDNF protein secreted by the positive cell strain constructed by pPBmL-PNC-TPA-Fc-GDNF is equivalent to that of BDNF protein secreted by the positive cell strain constructed by pPBmL-PNC-TPA-BDNF-Fc, and the difference is not large.
Example 6
Protein biological function assay
First with R&D and self-produced GDNF or BDNF are respectively prepared into a motor neuron and a dopaminergic neuron differentiation kit for verifying the biological functions of the GDNF or BDNF obtained by different methods. The differentiation steps were as follows: selecting hPSC cells with good growth, digesting with Solase cell digestive juice, counting Vi-cells, adjusting cell density according to 5 × 105cells/well and 2 mL/well are inoculated to a 6-well plate, different differentiation culture media are selected at different culture stages, and the detailed differentiation steps are described in the specification (the kit is prepared by Shanghai-Shenyu Biotech Co., Ltd. in Anhui). And (5) until the cells grow to the mature stage, photographing to observe the cell morphology, and recording the cell characteristics. After the photographing is finished, the Solase cell digestive juice is digested, cell sediment is collected by centrifugation, and the expression condition of the Marker with the specificity of the motor neuron and the dopaminergic neuron is detected by a QPCR detection kit (manufacturer: Beijing Quanjin, cat number: AQ 131-04).
The cell picture and QPCR detection results are shown in fig. 12, in which fig. 12A shows the motor neuron cell morphology and Marker expression, and fig. 12B shows the dopaminergic neuron cell morphology and Marker expression. The results show that R & D and the motor neurons and dopaminergic neurons obtained by the GDNF differentiation culture of the invention have no significant difference in cell morphology and show respective unique fibrous structures and cell morphologies; the expression of the Marker has no obvious difference, and the Marker markers which are respectively unique are expressed, so that the expression level is high and has no difference. The use concentration of R & D GDNF is 10ng/ml in the experimental differentiation process, the use concentration of the GDNF is 8ng/ml, the result can be obtained, the lower use amount achieves the same effect, and the activity of the GDNF expressed by the recombination has obvious advantages.
As can be seen from the results of the above examples, the present invention obtains dimeric GDNF or BDNF with high biological activity by constructing a high expression vector and screening high expression cell lines using a cell expression system. The method has the advantages of simple operation, high yield, simple and time-saving purification, high purity of the obtained dimeric GDNF or BDNF, high purity of endotoxin meeting the clinical use requirement, production cost reduction, solving the defects of low expression level, high price, low biological activity and the like of the recombinant GDNF or BDNF on the market, and being beneficial to large-scale production, process amplification, application of relevant cells and diseases such as motor neurons and the like of the GDNF or BDNF protein.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Shang Sheng Yuan Biotechnology Co., Ltd in Anhui
<120> method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application thereof
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Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
355 360 365
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
370 375 380
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
385 390 395 400
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
405 410 415
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
420 425 430
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
435 440 445
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
450 455 460
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
465 470 475 480
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
485 490 495
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
500 505 510
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
515 520 525
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
530 535 540
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
545 550 555 560
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
565 570 575
Ser Leu Ser Leu Ser Pro Gly Lys
580
<210> 3
<211> 23
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser
20
<210> 4
<211> 231
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
1 5 10 15
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
20 25 30
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
35 40 45
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
50 55 60
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
65 70 75 80
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
85 90 95
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
100 105 110
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
115 120 125
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
130 135 140
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
145 150 155 160
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
165 170 175
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
180 185 190
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
195 200 205
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
210 215 220
Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 5
<211> 391
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Pro Lys Ser Cys Asp Lys Thr His Thr
20 25 30
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
35 40 45
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
50 55 60
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
65 70 75 80
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
85 90 95
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
100 105 110
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
115 120 125
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
130 135 140
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
145 150 155 160
Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
165 170 175
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
180 185 190
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
195 200 205
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
210 215 220
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
225 230 235 240
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Thr Arg
245 250 255
Met Ser Pro Asp Lys Gln Met Ala Val Leu Pro Arg Arg Glu Arg Asn
260 265 270
Arg Gln Ala Ala Ala Ala Asn Pro Glu Asn Ser Arg Gly Lys Gly Arg
275 280 285
Arg Gly Gln Arg Gly Lys Asn Arg Gly Cys Val Leu Thr Ala Ile His
290 295 300
Leu Asn Val Thr Asp Leu Gly Leu Gly Tyr Glu Thr Lys Glu Glu Leu
305 310 315 320
Ile Phe Arg Tyr Cys Ser Gly Ser Cys Asp Ala Ala Glu Thr Thr Tyr
325 330 335
Asp Lys Ile Leu Lys Asn Leu Ser Arg Asn Arg Arg Leu Val Ser Asp
340 345 350
Lys Val Gly Gln Ala Cys Cys Arg Pro Ile Ala Phe Asp Asp Asp Leu
355 360 365
Ser Phe Leu Asp Asp Asn Leu Val Tyr His Ile Leu Arg Lys His Ser
370 375 380
Ala Lys Arg Cys Gly Cys Ile
385 390
<210> 6
<211> 1176
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgcccagcc caaagagctg cgacaagaca cacacctgcc caccatgtcc agcacctgag 120
ctgctgggag gaccaagcgt gttcctgttt cctccaaagc ccaaggatac actgatgatc 180
tctaggaccc ctgaggtgac atgcgtggtg gtggacgtga gccacgaaga cccagaggtg 240
aagtttaact ggtacgtgga cggcgtggag gtgcacaatg ccaagaccaa gcccagggag 300
gagcagtaca actccaccta tcgcgtggtg tctgtgctga cagtgctgca ccaggattgg 360
ctgaacggca aggagtataa gtgtaaggtg tccaataagg ccctgccagc ccccatcgag 420
aagaccatct ctaaggcaaa gggacagcca agggagcctc aggtgtacac actgccccct 480
tccagagacg agctgaccaa gaaccaggtg tctctgacat gcctggtgaa gggcttctat 540
ccatccgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ttacaagacc 600
acaccacccg tgctggactc cgatggctct ttctttctgt attccaagct gaccgtggac 660
aagtctcggt ggcagcaggg caacgtgttt agctgctccg tgatgcacga ggccctgcac 720
aatcactaca cacagaagtc tctgagcctg tcccccggca agacgcgtat gagccccgat 780
aagcagatgg ccgtgctgcc tcggagagag aggaacaggc aggcagcagc agcaaaccca 840
gagaattcca ggggcaaggg caggcgcgga cagaggggca agaacagagg ctgcgtgctg 900
accgccatcc acctgaatgt gacagatctg ggcctgggct acgagaccaa ggaggagctg 960
atcttccggt attgcagcgg ctcctgtgat gccgccgaga ccacatacga caagatcctg 1020
aagaacctgt ctcggaatcg gagactggtg agcgacaaag tgggccaggc ctgctgtaga 1080
cccatcgcct tcgacgatga cctgtccttt ctggatgaca atctggtgta tcacatcctg 1140
aggaagcact ctgccaagcg gtgcggctgt atctga 1176
<210> 7
<211> 353
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Met His Ser Asp Pro Ala Arg Arg Gly Glu Leu Ser Val Cys Asp Ser
1 5 10 15
Ile Ser Glu Trp Val Thr Ala Ala Asp Lys Lys Thr Ala Val Asp Met
20 25 30
Ser Gly Gly Thr Val Thr Val Leu Glu Lys Val Pro Val Ser Lys Gly
35 40 45
Gln Leu Lys Gln Tyr Phe Tyr Glu Thr Lys Cys Asn Pro Met Gly Tyr
50 55 60
Thr Lys Glu Gly Cys Arg Gly Ile Asp Lys Arg His Trp Asn Ser Gln
65 70 75 80
Cys Arg Thr Thr Gln Ser Tyr Val Arg Ala Leu Thr Met Asp Ser Lys
85 90 95
Lys Arg Ile Gly Trp Arg Phe Ile Arg Ile Asp Thr Ser Cys Val Cys
100 105 110
Thr Leu Thr Ile Lys Arg Gly Arg Thr Arg Pro Lys Ser Cys Asp Lys
115 120 125
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
130 135 140
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
145 150 155 160
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
165 170 175
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
180 185 190
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
195 200 205
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
210 215 220
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
225 230 235 240
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
245 250 255
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
260 265 270
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
275 280 285
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
290 295 300
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
305 310 315 320
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
325 330 335
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
340 345 350
Lys
<210> 8
<211> 1131
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgcccagca tgcactccga cccagcaagg agaggagagc tgagcgtgtg cgatagcatc 120
tccgagtggg tgaccgccgc cgacaagaag acagccgtgg atatgagcgg cggcaccgtg 180
acagtgctgg agaaggtgcc cgtgtccaag ggccagctga agcagtactt ctatgagacc 240
aagtgcaacc ctatgggcta cacaaaggag ggctgtaggg gcatcgacaa gcgccactgg 300
aattctcagt gtcggaccac acagagctat gtgcgggccc tgaccatgga ctctaagaag 360
agaatcggct ggcggtttat cagaatcgat acatcctgcg tgtgcaccct gacaatcaag 420
aggggccgca cgcgtccaaa gagctgcgac aagacacaca cctgcccacc atgtccagca 480
cctgagctgc tgggaggacc aagcgtgttc ctgtttcctc caaagcccaa ggatacactg 540
atgatctcta ggacccctga ggtgacatgc gtggtggtgg acgtgagcca cgaagaccca 600
gaggtgaagt ttaactggta cgtggacggc gtggaggtgc acaatgccaa gaccaagccc 660
agggaggagc agtacaactc cacctatcgc gtggtgtctg tgctgacagt gctgcaccag 720
gattggctga acggcaagga gtataagtgt aaggtgtcca ataaggccct gccagccccc 780
atcgagaaga ccatctctaa ggcaaaggga cagccaaggg agcctcaggt gtacacactg 840
cccccttcca gagacgagct gaccaagaac caggtgtctc tgacatgcct ggtgaagggc 900
ttctatccat ccgatatcgc cgtggagtgg gagtctaatg gccagcccga gaacaattac 960
aagaccacac cacccgtgct ggactccgat ggctctttct ttctgtattc caagctgacc 1020
gtggacaagt ctcggtggca gcagggcaac gtgtttagct gctccgtgat gcacgaggcc 1080
ctgcacaatc actacacaca gaagtctctg agcctgtccc ccggcaagtg a 1131
<210> 9
<211> 69
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgcccagc 69
<210> 10
<211> 693
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ccaaagagct gcgacaagac acacacctgc ccaccatgtc cagcacctga gctgctggga 60
ggaccaagcg tgttcctgtt tcctccaaag cccaaggata cactgatgat ctctaggacc 120
cctgaggtga catgcgtggt ggtggacgtg agccacgaag acccagaggt gaagtttaac 180
tggtacgtgg acggcgtgga ggtgcacaat gccaagacca agcccaggga ggagcagtac 240
aactccacct atcgcgtggt gtctgtgctg acagtgctgc accaggattg gctgaacggc 300
aaggagtata agtgtaaggt gtccaataag gccctgccag cccccatcga gaagaccatc 360
tctaaggcaa agggacagcc aagggagcct caggtgtaca cactgccccc ttccagagac 420
gagctgacca agaaccaggt gtctctgaca tgcctggtga agggcttcta tccatccgat 480
atcgccgtgg agtgggagtc taatggccag cccgagaaca attacaagac cacaccaccc 540
gtgctggact ccgatggctc tttctttctg tattccaagc tgaccgtgga caagtctcgg 600
tggcagcagg gcaacgtgtt tagctgctcc gtgatgcacg aggccctgca caatcactac 660
acacagaagt ctctgagcct gtcccccggc aag 693
<210> 11
<211> 1926
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgcccagcc caaagagctg cgacaagaca cacacctgcc caccatgtcc agcacctgag 120
ctgctgggag gaccaagcgt gttcctgttt cctccaaagc ccaaggatac actgatgatc 180
tctaggaccc ctgaggtgac atgcgtggtg gtggacgtga gccacgaaga cccagaggtg 240
aagtttaact ggtacgtgga cggcgtggag gtgcacaatg ccaagaccaa gcccagggag 300
gagcagtaca actccaccta tcgcgtggtg tctgtgctga cagtgctgca ccaggattgg 360
ctgaacggca aggagtataa gtgtaaggtg tccaataagg ccctgccagc ccccatcgag 420
aagaccatct ctaaggcaaa gggacagcca agggagcctc aggtgtacac actgccccct 480
tccagagacg agctgaccaa gaaccaggtg tctctgacat gcctggtgaa gggcttctat 540
ccatccgata tcgccgtgga gtgggagtct aatggccagc ccgagaacaa ttacaagacc 600
acaccacccg tgctggactc cgatggctct ttctttctgt attccaagct gaccgtggac 660
aagtctcggt ggcagcaggg caacgtgttt agctgctccg tgatgcacga ggccctgcac 720
aatcactaca cacagaagtc tctgagcctg tcccccggca agaagagagg gctctgctgt 780
gtgctgctgc tgtgtggagc agtcttcgtt tcgcccagcc caaagagctg cgacaagaca 840
cacacctgcc caccatgtcc agcacctgag ctgctgggag gaccaagcgt gttcctgttt 900
cctccaaagc ccaaggatac actgatgatc tctaggaccc ctgaggtgac atgcgtggtg 960
gtggacgtga gccacgaaga cccagaggtg aagtttaact ggtacgtgga cggcgtggag 1020
gtgcacaatg ccaagaccaa gcccagggag gagcagtaca actccaccta tcgcgtggtg 1080
tctgtgctga cagtgctgca ccaggattgg ctgaacggca aggagtataa gtgtaaggtg 1140
tccaataagg ccctgccagc ccccatcgag aagaccatct ctaaggcaaa gggacagcca 1200
agggagcctc aggtgtacac actgccccct tccagagacg agctgaccaa gaaccaggtg 1260
tctctgacat gcctggtgaa gggcttctat ccatccgata tcgccgtgga gtgggagtct 1320
aatggccagc ccgagaacaa ttacaagacc acaccacccg tgctggactc cgatggctct 1380
ttctttctgt attccaagct gaccgtggac aagtctcggt ggcagcaggg caacgtgttt 1440
agctgctccg tgatgcacga ggccctgcac aatcactaca cacagaagtc tctgagcctg 1500
tcccccggca agacgcgtat gagccccgat aagcagatgg ccgtgctgcc tcggagagag 1560
aggaacaggc aggcagcagc agcaaaccca gagaattcca ggggcaaggg caggcgcgga 1620
cagaggggca agaacagagg ctgcgtgctg accgccatcc acctgaatgt gacagatctg 1680
ggcctgggct acgagaccaa ggaggagctg atcttccggt attgcagcgg ctcctgtgat 1740
gccgccgaga ccacatacga caagatcctg aagaacctgt ctcggaatcg gagactggtg 1800
agcgacaaag tgggccaggc ctgctgtaga cccatcgcct tcgacgatga cctgtccttt 1860
ctggatgaca atctggtgta tcacatcctg aggaagcact ctgccaagcg gtgcggctgt 1920
atctga 1926
<210> 12
<211> 1893
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60
tcgcccagca tggatgcaat gaagagaggg ctctgctgtg tgctgctgct gtgtggagca 120
gtcttcgttt cgcccagcat gcactccgac ccagcaagga gaggagagct gagcgtgtgc 180
gatagcatct ccgagtgggt gaccgccgcc gacaagaaga cagccgtgga tatgagcggc 240
ggcaccgtga cagtgctgga gaaggtgccc gtgtccaagg gccagctgaa gcagtacttc 300
tatgagacca agtgcaaccc tatgggctac acaaaggagg gctgtagggg catcgacaag 360
cgccactgga attctcagtg tcggaccaca cagagctatg tgcgggccct gaccatggac 420
tctaagaaga gaatcggctg gcggtttatc agaatcgata catcctgcgt gtgcaccctg 480
acaatcaaga ggggccgcac gcgtccaaag agctgcgaca agacacacac ctgcccacca 540
tgtccagcac ctgagctgct gggaggacca agcgtgttcc tgtttcctcc aaagcccaag 600
gatacactga tgatctctag gacccctgag gtgacatgcg tggtggtgga cgtgagccac 660
gaagacccag aggtgaagtt taactggtac gtggacggcg tggaggtgca caatgccaag 720
accaagccca gggaggagca gtacaactcc acctatcgcg tggtgtctgt gctgacagtg 780
ctgcaccagg attggctgaa cggcaaggag tataagtgta aggtgtccaa taaggccctg 840
ccagccccca tcgagaagac catctctaag gcaaagggac agccaaggga gcctcaggtg 900
tacacactgc ccccttccag agacgagctg accaagaacc aggtgtctct gacatgcctg 960
gtgaagggct tctatccatc cgatatcgcc gtggagtggg agtctaatgg ccagcccgag 1020
aacaattaca agaccacacc acccgtgctg gactccgatg gctctttctt tctgtattcc 1080
aagctgaccg tggacaagtc tcggtggcag cagggcaacg tgtttagctg ctccgtgatg 1140
cacgaggccc tgcacaatca ctacacacag aagtctctga gcctgtcccc cggcaagtga 1200
ccaaagagct gcgacaagac acacacctgc ccaccatgtc cagcacctga gctgctggga 1260
ggaccaagcg tgttcctgtt tcctccaaag cccaaggata cactgatgat ctctaggacc 1320
cctgaggtga catgcgtggt ggtggacgtg agccacgaag acccagaggt gaagtttaac 1380
tggtacgtgg acggcgtgga ggtgcacaat gccaagacca agcccaggga ggagcagtac 1440
aactccacct atcgcgtggt gtctgtgctg acagtgctgc accaggattg gctgaacggc 1500
aaggagtata agtgtaaggt gtccaataag gccctgccag cccccatcga gaagaccatc 1560
tctaaggcaa agggacagcc aagggagcct caggtgtaca cactgccccc ttccagagac 1620
gagctgacca agaaccaggt gtctctgaca tgcctggtga agggcttcta tccatccgat 1680
atcgccgtgg agtgggagtc taatggccag cccgagaaca attacaagac cacaccaccc 1740
gtgctggact ccgatggctc tttctttctg tattccaagc tgaccgtgga caagtctcgg 1800
tggcagcagg gcaacgtgtt tagctgctcc gtgatgcacg aggccctgca caatcactac 1860
acacagaagt ctctgagcct gtcccccggc aag 1893

Claims (17)

1. A fusion gene for coding recombinant neurotrophic factors, which is characterized by comprising a neurotrophic factor gene sequence, a secretion signal peptide TPA gene sequence and an Fc gene sequence.
2. The fusion gene of claim 1, wherein the fusion gene of the recombinant neurotrophic factor is a secretion signal peptide TPA gene sequence-Fc gene sequence-neurotrophic factor gene sequence or a secretion signal peptide TPA gene sequence-neurotrophic factor gene sequence-Fc gene sequence.
3. The fusion gene of claim 2, wherein the neurotrophic factor is GDNF or BDNF;
the nucleotide sequence of the GDNF is shown as SEQ ID NO. 6, and the nucleotide sequence of the BDNF is shown as SEQ ID NO. 8.
4. The fusion gene of claim 3, wherein the TPA gene sequence of the secretion signal peptide is shown in SEQ ID NO. 9, and the Fc gene sequence is shown in SEQ ID NO. 10.
5. The fusion gene of claim 4, wherein the fusion gene of the recombinant GDNF is TPA gene sequence-Fc gene sequence-GDNF gene sequence, and the nucleotide sequence is shown in SEQ ID NO: 11;
the fusion gene of the recombinant BDNF is a TPA gene sequence-BDNF gene sequence-Fc gene sequence, and the nucleotide sequence is shown in SEQ ID NO. 12.
6. A recombinant expression vector comprising the fusion gene according to any one of claims 1 to 5.
7. The recombinant expression vector of claim 6, wherein the backbone vector is pPBml-PNCE.
8. A recombinant cell comprising the fusion gene according to any one of claims 1 to 5 or the recombinant expression vector according to claim 6 or 7.
9. The recombinant cell of claim 8, wherein the recombinant cell is a HEK293 cell.
10. A method of preparing a recombinant neurotrophin fusion protein, comprising the steps of:
1) constructing a recombinant expression vector containing the fusion gene of any one of claims 1 to 5;
2) transforming the recombinant expression vector in the step 1) into a mammalian cell, screening a high-expression cell strain from the obtained recombinant cell, culturing, separating supernatant, and purifying to obtain the recombinant protein.
11. The method as claimed in claim 10, wherein the method for screening high expression cell lines comprises detecting the expression level of the fusion protein by an Anti-Fc Elisa detection kit, and selecting a cell line with the highest protein expression level for culture.
12. The method of claim 10, wherein the purification is performed by Protein A column, and the Loading buffer containing Igepal CA-630 is used to remove endotoxin from the Protein.
13. The method of claim 12, wherein the content of the Igepal CA-630 is 0.1-2% by mass.
14. The method according to claim 12 or 13, wherein the flow rate of the Loading buffer containing Igepal CA-630 is 0.4-2 mL/min.
15. A fusion protein expressed by the recombinant cell produced by the method of any one of claims 10 to 14, wherein the fusion protein is a dimer; the fusion protein comprises a neurotrophic factor and an Fc structure, wherein the neurotrophic factor is GDNF or BDNF.
16. Use of the recombinant neurotrophin fusion protein of claim 15 for promoting neuronal cell growth and/or differentiation.
17. The use of claim 16, wherein the neurons comprise dopaminergic neurons and/or motor neurons.
CN202111363112.XA 2021-11-17 2021-11-17 Method for expressing recombinant neurotrophic factor fusion protein, recombinant neurotrophic factor fusion protein and application thereof Pending CN114075569A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302529A (en) * 2007-05-08 2008-11-12 王尚武 Transgenic neural stem cell co-expressing GDNF and BDNF
CN108300736A (en) * 2016-09-12 2018-07-20 中国食品药品检定研究院 The Chinese hamster ovary celI strain of efficiently expressing recombinant human β-NGF-Fc fusion proteins and its construction method
CN108623695A (en) * 2017-03-24 2018-10-09 中国科学院过程工程研究所 A kind of albumin binding peptide-human ciliary nerve nutrition factor fusion protein and its preparation method and application
CN110713983A (en) * 2019-11-04 2020-01-21 深圳市体内生物医药科技有限公司 Immune cell for expressing hyaluronidase and application thereof
WO2021138350A1 (en) * 2019-12-29 2021-07-08 Gloriana Therapeutics Mammalian cells secreting gdnf and their therapeutic use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302529A (en) * 2007-05-08 2008-11-12 王尚武 Transgenic neural stem cell co-expressing GDNF and BDNF
CN108300736A (en) * 2016-09-12 2018-07-20 中国食品药品检定研究院 The Chinese hamster ovary celI strain of efficiently expressing recombinant human β-NGF-Fc fusion proteins and its construction method
CN108623695A (en) * 2017-03-24 2018-10-09 中国科学院过程工程研究所 A kind of albumin binding peptide-human ciliary nerve nutrition factor fusion protein and its preparation method and application
CN110713983A (en) * 2019-11-04 2020-01-21 深圳市体内生物医药科技有限公司 Immune cell for expressing hyaluronidase and application thereof
WO2021138350A1 (en) * 2019-12-29 2021-07-08 Gloriana Therapeutics Mammalian cells secreting gdnf and their therapeutic use

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
吕世静: "临床免疫学检验", 中国医药科技出版社, pages: 38 *
蒋晖;金大地;鲁凯伍;金卫林;鞠躬;: "新型神经营养因子TAT―BDNF生物合成及生物活性研究", 中国临床解剖学杂志, no. 03 *
高小青;周玲;杜杰;杨朝鲜;: "GDNF基因修饰的BMSCs对大鼠缺氧复氧神经细胞凋亡的实验研究", 重庆医学, no. 13 *

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