Recombinant botulinum toxin and preparation method thereof

Abstract

The present invention relates to a recombinant botulinum toxin and a preparation method thereof, and relates to the technical field of gene engineering, the recombinant botulinum toxin comprises an antibody light chain variable region as a tag protein, and the protein sequence of the antibody light chain variable region is represented by SEQ ID NO: 3. The tag protein used in the method is moderate in size and simple in structure, can be subjected to affinity purification by using Protein L filler, and is high in yield and good in specificity. Especially, elution of affinity purification can be realized only through low pH, and enzyme digestion can be carried out after an eluent is adjusted to a proper pH value. Moreover, the operations of removing an eluent through ultrafiltration and the like are not needed, the enzyme digestion product can be subjected to affinity purification again, tag protein and related impurities are efficiently removed, the production steps and cost of the botulinum toxin are greatly simplified, and the defects in the prior art are overcome.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a recombinant botulinum toxin and a preparation method thereof. Background Art

[0002] Botulinum toxin, also known as botulinum toxin, is a neurotoxin protein produced by Clostridium botulinum during its reproduction process. Botulinum toxin is divided into seven immune serum subtypes, A to G, according to its toxicity and antigenicity. Different subtypes exist in a double-chain form, consisting of a heavy chain and a light chain connected by a disulfide bond. The heavy chain can bind to specific receptors on the outer surface of nerve ending cells and assist the light chain to enter the cell through binding-transport. Inside the cell, the light and heavy chains dissociate and the light chain is released. The light chain has zinc endopeptidase activity, which specifically cuts the related proteins in neurons responsible for the fusion of vesicles and cell membranes. BoNT / A, BoNT / C and BoNT / E cleave 25kDa synaptosome-associated protein (SNAP25), BoNT / B, BoNT / D, BoNT / F and BoNT / G cleave synaptic vesicle membrane protein (VAMP), and BoNT / C cleaves SNAP25 and syntaxin, thereby blocking the release of the neurotransmitter acetylcholine, causing the muscles to lose their innervation, resulting in reduced muscle tone and relief of muscle spasms. Based on the property of causing muscle relaxation, botulinum toxin has long been used in beauty and disease treatment, including bladder hyperactivity, hyperhidrosis, cervical dystonia, blepharospasm, frown lines, etc.

[0003] At present, all botulinum toxins used for treatment are natural botulinum toxins, which are obtained by culturing, separating and purifying Clostridium botulinum. The preparation process has high requirements for plant and production equipment, and the risk of pollution to the environment and personnel is also relatively high. In addition, the prepared botulinum toxin is a complex with a molecular weight of 300-900kDa. In addition to botulinum toxin protein, it also contains lectin protein and non-toxin non-hemagglutinin protein. This complex will produce an immune response after long-term use, resulting in a weakened effect or causing other side effects.

[0004] Through genetic engineering methods, the light and heavy chains of botulinum toxin are fused and expressed, and protease cleavage sites are designed between the light and heavy chains. After the fusion protein is purified, it is cleaved with protease to obtain active protein, which can make up for the shortcomings of extracting botulinum toxin from Clostridium botulinum. First, the fusion protein that has not been activated by protease has very low toxicity. The early cultivation and purification production process does not require special plant equipment and has a high safety factor. Secondly, the recombinantly expressed botulinum toxin is highly purified, does not contain non-toxin components, and has more uniform composition and physical and chemical properties. After industrialization, the consistency between batches will be better, and in theory, clinical safety will also be better. Many patents have disclosed this technical route and successfully prepared active botulinum toxin, such as Xuan Wei used insect cells to express BoNT / A with a His tag (Xuan Wei et al., 2023); CN115785237 fused the His tag to the heavy chain, and the fusion sequence and the light chain were co-expressed in Escherichia coli; WO2006076902 expressed BoNT / A soluble in Escherichia coli; WO2019101308 expressed BoNT / A with a His tag in Escherichia coli and activated it with thrombin; WO0114570 disclosed a variety of schemes for fusing botulinum toxin with a His tag, GST tag, MBP tag or CBP tag, and activating it with trypsin or ArgC protease; CN202110217647 used GST expression and activated it with 3C protease; CN116813727 simultaneously fused the His tag and SUMO tag to the N-terminus of BoNT / A and activated it with thrombin. It can be seen from the above literature that the current preparation of recombinant botulinum toxin mostly uses Escherichia coli as the host bacteria, and the method of fusion expression of botulinum toxin and affinity-purifiable tag protein. In this method, the selection and design of the tag protein is crucial. On the one hand, the tag protein cannot affect the structure of the target botulinum toxin protein after fusion, so as to avoid reducing the activity of the botulinum toxin protein; on the other hand, the tag protein must be specifically and highly combined with the affinity filler to reduce the difficulty of purifying the botulinum toxin protein and improve the purity of the product; thirdly, the tag protein needs to be cut off at the same time as the botulinum toxin is activated by protease, and further separated and removed, otherwise it will affect the purity of the botulinum toxin product. Based on these goals, the present invention uses the antibody light chain variable region as the tag protein and fusion expression to prepare botulinum toxin. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a recombinant botulinum toxin and a preparation method thereof.

[0006] In a first aspect, the present invention provides a recombinant botulinum toxin, wherein the recombinant botulinum toxin comprises an antibody light chain variable region as a tag protein, and the protein sequence of the antibody light chain variable region is shown in SEQ ID NO:3.

[0007] Furthermore, the DNA sequence encoding the antibody light chain variable region is shown in SEQ ID NO:4.

[0008] Furthermore, the botulinum toxin includes botulinum toxin type A, and the amino acid sequence of the botulinum toxin type A is shown in SEQ ID NO:1.

[0009] Furthermore, the DNA sequence encoding the botulinum toxin type A is shown in SEQ ID NO:2.

[0010] Furthermore, in the amino acid sequence shown in SEQ ID NO: 1, 1-435 is the light chain of the type A botulinum toxin, 438-443 is the enterokinase recognition cleavage site, and 446-1294 is the heavy chain of the type A botulinum toxin.

[0011] Furthermore, the antibody light chain variable region-botulinum toxin fusion protein sequence is shown in SEQ ID NO:5.

[0012] Furthermore, the nucleotide sequence of the antibody light chain variable region-botulinum toxin fusion protein is shown in SEQ ID NO:6.

[0013] In a second aspect, the present invention provides a method for preparing the recombinant botulinum toxin according to any one of the first aspects, the preparation method comprising the following steps:

[0014] Using the variable region of the antibody light chain as a tag protein, fusion expression is used to prepare recombinant botulinum toxin;

[0015] The recombinant botulinum toxin is purified using Protein L filler.

[0016] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0017] The present invention provides a recombinant botulinum toxin and a method for preparing the same. The tag protein used in the present invention is of moderate size and simple structure, and can be affinity purified using Protein L filler with high yield and good specificity. In particular, affinity purification elution can be achieved only by low pH, and enzyme cleavage can be performed after the eluent is adjusted to an appropriate pH. In addition, the enzyme cleavage product can be affinity purified again without the need for ultrafiltration to remove the eluent, and the tag protein and related impurities can be removed efficiently, which greatly simplifies the production steps and costs of botulinum toxin and makes up for the deficiencies of the prior art. Specifically:

[0018] 1) The antibody light chain variable region and Protein L filler have high loading capacity, strong affinity and good specificity, and the target protein with a purity of more than 90% can be obtained in one step of purification.

[0019] 2) The molecular weight of the variable region of the antibody light chain is about 13 kDa, which is of moderate size. It contains two cysteines, forming a pair of disulfide bonds, and no free cysteines, which reduces the probability of mismatch between the cysteine ​​in the tag and the cysteine ​​in the target protein.

[0020] 3) When using the His tag for purification, imidazole elution is required, the GST tag needs to be eluted with reduced glutathione, and the MBP tag needs to be eluted with maltose. The present invention uses the variable region of the antibody light chain as a tag and purifies with ProteinL filler, and elution can be achieved only by low pH, reducing production costs. At the same time, the buffer composition and salt concentration of the eluent do not change significantly, and there are no redundant components except the buffer system, which has no effect on the subsequent enzyme cleavage reaction and the stability of the botulinum toxin protein. Enzyme cleavage is performed under neutral and alkaline conditions, and the enzyme cleavage solution can be directly purified with ProteinL filler, and the cut label and the components with the label can be re-bound to the filler without being interfered by the salt concentration, and there is no need to add steps to remove eluents such as imidazole, reduced glutathione, and maltose, which greatly simplifies the operation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0023] Figure 1 This is a diagram showing the fusion expression and purification results using histidine or MBP as tags in an example of the present invention.

[0024] Figure 2 Schematic diagram of the fusion protein structure in the embodiment of the present invention.

[0025] Figure 3 This is a diagram showing the expression and purification results of the antibody light chain variable region in an embodiment of the present invention.

[0026] Figure 4 This is a diagram showing the expression and purification results of the light chain variable region-botulinum toxin fusion protein in an example of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0029] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, the conventional conditions disclosed in the prior art such as those mentioned in the background, or the conditions recommended by the manufacturer are carried out.

[0030] The sequences involved in the following examples are as follows:

[0031] SEQ ID NO: 1 is as follows:

[0032]

[0033] SEQ ID NO:2 is as follows:

[0034] TTTGTTAATAAACAGTTCAACTACAAGGACCCGGTTAATGGCGTGGATATTGCC

[0035] TATATTAAGATCCCGAATGTGGGCCAGATGCAGCCGGTTAAAGCCTTTAAAATTCAT

[0036] AACAAGATCTGGGTGATCCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGCGA

[0037] TCTGAATCCGCCGCCTGAAGCCAAACAGGTGCCGGTGTCATATTATGATAGTACCTA

[0038] TCTGAGCACCGATAATGAAAAAGATAACTACCTGAAGGGCGTGACCAAACTGTTTG

[0039] AACGCATTTATAGCACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTGCGCGGCA

[0040] TTCCGTTTTGGGGTGGCTCAACCATTGATACCGAACTGAAAGTTATCGATACCAATT

[0041] GCATCAACGTGATTCAGCCGGATGGTAGTTATCGCAGTGAAGAACTGAATCTGGTT

[0042] ATTATTGGCCCGAGCGCCGATATTATTCAGTTTGAATGTAAGAGCTTCGGCCATGAA

[0043] GTGCTGAATCTGACCCGTAATGGTTATGGCAGCACCCAGTATATTCGCTTTAGTCCG

[0044] GATTTTACCTTCGGTTTTGAAGAAAGCCTGGAAGTTGATACCAATCCGCTGCTGGGT

[0045] GCAGGTAAATTTGCAACCGATCCGGCCGTGACCCTGGCTCACGAGTTAATTCATGCC

[0046] GGTCATCGCCTGTATGGCATTGCAATTAATCCGAATCGTGTTTTTAAGGTGAACACC

[0047] AATGCATATTACGAGATGAGCGGTCTGGAAGTTAGCTTTGAAGAACTGCGTACCTTT

[0048] GGTGGTCATGATGCCAAATTTATTGACAGTCTGCAGGAAAATGAGTTCCGCCTGTAT

[0049] TATTATAACAAGTTCAAGGACATCGCAAGCACCCTGAATAAAGCCAAAAGCATTGT

[0050] GGGTACCACCGCAAGCCTGCAGTATATGAAAAATGTTTTCAAGGAGAAGTACCTGC

[0051] TGAGTGAAGATACCAGCGGTAAATTTAGTGTTGATAAGCTGAAGTTCGACAAGCTG

[0052] TATAAAATGCTGACCGAAATTTACACCGAGGATAATTTTGTGAAGTTCTTCAAGGTG

[0053] CTGAACCGTAAAACCTATCTGAATTTTGACAAGGCAGTGTTTAAGATCAACATCGTG

[0054] CCGAAAGTGAATTATACCATTTACGATGGCTTCAACCTGCGCAATACCAATCTGGCA

[0055] GCCAATTTTAATGGTCAGAATACCGAAATCAACAACATGAATTTCACCAAGCTGAA

[0056] GAACTTCACCGGTCTGTTTGAATTTTACAAGCTGCTGTGCGTTCGTGGTATTATTACC

[0057] AGTGGTGGTAGCGATGATGATGATAAAGGCGGTAGTGCACTGAATGATCTGTGTAT

[0058] TAAAGTGAACAACTGGGATCTGTTCTTCAGTCCGAGCGAAGATAATTTTACCAATGA

[0059] TCTGAACAAGGGCGAAGAAATTACCAGCCGATACCAATATTGAGGCCGCAGAAGAA

[0060] AATATTAGCCTGGATCTGATTCAGCAGTATTATCTGACCTTTAACTTCGATAACGAG

[0061] CCGGAAAATATCAGTATTGAAAACCTGAGTAGCGACATTATCGGTCAGCTGGAACT

[0062] GATGCCGAATATTGAACGCTTTCCGAATGGTAAAAGTACGAACTGGATAAATACA

[0063] CCATGTTCCATTACCTGCGCGCACAGGAATTTGAACATGGCAAAGCCGCATTGCA

[0064] CTGACCAATAGTGTGAAATGAAGCACTGCTGAATCCGAGCCGCGTGTATACCTTTTTT

[0065] AGTAGTGATTACGTGAAGAAGGTGAACAAAGCCACCGAAGCAGCAATGTTTCTGGG

[0066] CTGGGTTGAACAGCTGGTTTATGATTTTACCGATGAAACCAGCGAAGTTAGCACCAC

[0067] CGATAAAATTGCCGATATTACCATTATCCCCGTATATTGGCCCGGCACTGAATAT

[0068] TGGCAATATGCTGTATAAGGACGACTTTGTGGGCGCCCTGATTTTTAGCGGCGCCGT

[0069] TATTCTGCTGGAATTTATTCCGGAAATCGCCATTCCGGTGCTGGGTACCTTTGCACT

[0070] GGTTAGTTATATTGCCAATAAGGTGCTGACCGTTCAGACCATTGATAATGCACTGAG

[0071] CAAACGCAATGAAAAATGGGATGAAGTTTACAAGTACATCGTGACCAATTGGCTGG

[0072] CCAAAGTTAATACCCAGATTGATCTGATTCGCAAAAAGATGAAGGAGGCACTGGAA

[0073] AATCAGGCCGAAGCAACCAAAGCAATTATTAATTACCAGTACAACCAGTACACCGA

[0074] AGAAGAAAAAAACAACATCAACTTCAACATCGACGACCTGAGCAGCAAACTGAAT

[0075] GAAAGTATTAACAAGGCCATGATCAACATCAACAAGTTTCTGAATCAGTGCAGTGT

[0076] GAGTTATCTGATGAATAGCATGATCCCGTATGGTGTTAAACGTCTGGAAGATTTTGA

[0077] CGCCAGCCTGAAAGATGCCCTGCTGAAATATATTTACGACAATCGTGGCACCCTGAT

[0078] TGGTCAGGTGGATCGTCTGAAAGATAAAGTTAATAACACCCTGAGCACCGACATTC

[0079] CGTTTCAGCTGAGCAAATATGTTGATAATCAGCGCCTGCTGAGTACCTTTACCGAAT

[0080] ATATTAAGAACATCATCAACACCAGCATCCTGAATCTGCGTTATGAAAGCAATCATC

[0081] TGATCGATCTGAGCCGTTATGCCAGCAAAATTAATATTGGTAGCAAGGTTAACTTCG

[0082] ACCCGATTGATAAAAACCAGATTCAGCTGTTTAACCTGGAAAGCAGTAAAATTGAG

[0083] GTGATTCTGAAGAACGCAATCGTTTATAACAGCATGTACGAAAACTTCAGCACCAG

[0084] TTTTTGGATTCGCATTCCGAAATATTTCAACAGCATCAGTCTGAATAACGAGTATAC

[0085] CATTATCAACTGCATGGAAAACAACAGTGGCTGGAAAGTTAGCCTGAATTATGGCG

[0086] AAATTATCTGGACCCTGCAGGATACCCAGGAAATTAAACAGCGTGTGGTTTTTAAGT

[0087] ACAGCCAGATGATTAACATCAGCGATTATATCAACCGCTGGATTTTTGTGACCATTA

[0088] CAAATAACCGCCTGAATAATAGCAAGATCTACATTAACGGCCGCCTGATTGATCAG

[0089] AAACCGATTAGTAATCTGGGTAATATCCACGCAAGCAATAATATTATGTTCAAGCTG

[0090] GACGGTTGCCGTGATACCCATCGTTATATTTGGATTAAGTACTTCAACCTGTTCGAC

[0091] AAAGAGCTGAATGAAAAAGAAATCAAGGACCTGTACGACAATCAGAGTAATAGCG

[0092] GTATTCTGAAAGACTTTTGGGGTGATTATCTGCAGTATGATAAGCCGTATTACATGC

[0093] TGAATCTGTATGATCCGAACAAATACGTTGACGTTAACAATGTGGGTATCCGTGGTT

[0094] ATATGTATCTGAAAGGTCCGCGCGGTAGCGTTATGACCACCAATATTTATCTGAACA

[0095] GCAGCCTGTATCGCGGTACCAAATTTATTATTAAGAAGTACGCAAGCGGTAACAAG

[0096] GATAATATTGTTCGTAACAACGACCGCGTTTATATTAATGTTGTGGTTAAGAACAAG

[0097] GAGTACCGCCTGGCCACCAATGCAAGCCAGGCTGGTGTTGAAAAAATTCTGAGTGC

[0098] ACTGGAAATCCCGGATGTGGGCAATCTGAGTCAGGTTGTTGTTATGAAAAGCAAAA

[0099] ACGACCAGGGTATTACCAATAAATGCAAAATGAACCTGCAGGATAACAACGGCAAT

[0100] GATATTGGCTTTATCGGCTTTCATCAGTTTAACAATATCGCAAAGCTGGTGGCCAGT

[0101] AATTGGTATAATCGCCAGATTGAACGTAGCAGTCGTACCCTGGGTTGCAGTTGGGAATTTATTCCTGTGGATGATGGTTGGGGTGAACGCCCGCTGTGA。

[0102] SEQ ID NO:3 is as follows:

[0103] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESG VPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEI。

[0104] SEQ ID NO:4 is as follows:

[0105] GACATCCAGATGACCCAGAGCCCGAGTAGCCTGAGTGCAAGTGTGGGCGATCG

[0106] TGTGACCATTACCTGTAAAGCAAGCCGTAATATTGAGCGTCAGCTGGCATGGTATCA

[0107] GCAGAAACCGGGCCAGGCACCGGAATTACTGATTTATCAGGCAAGTCGCAAAGAA

[0108] AGCGGCGTGCCGGATCGCTTTAGCGGTAGTCGTTATGGTACCGATTTTACCCTGACC

[0109] ATTAGTAGTCTGCAGCCGGAAGATATTGCCACCTATTATTGTCAGCAGTATAGTGATCCGCCGCTGACCTTTGGCGGTGGTACAAAAGTTGAAATTTGA。

[0110] SEQ ID NO:5 is as follows:

[0111] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESG

[0112] VPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIGGSGGSGGSDD

[0113] DDDKGGSGGSFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTF

[0114] TNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSI

[0115] VRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLN

[0116] LTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYG

[0117] IAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIA

[0118] STLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDN

[0119] FVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMN

[0120] FTKLKNFTGLFEFYKLLCVRGIITSGGSDDDDKGGSALNDLCIKVNNWDLFFSPSEDNFT

[0121] NDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIE

[0122] RFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKV

[0123] NKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVG

[0124] ALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTN

[0125] WLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNE

[0126] SINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQV

[0127] DRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYAS

[0128] KINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLN

[0129] NEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTIT

[0130] NNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNE

[0131] KEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLK

[0132] GPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATN

[0133] ASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL。

[0134] SEQ ID NO:6 is as follows:

[0135] GACATCCAGATGACCCAGAGCCCGAGTAGCCTGAGTGCAAGTGTGGGCGATCG

[0136] TGTGACCATTACCTGTAAAGCAAGCCGTAATATTGAGCGTCAGCTGGCATGGTATCA

[0137] GCAGAAACCGGGCCAGGCACCGGAATTACTGATTTATCAGGCAAGTCGCAAAGAA

[0138] AGCGGCGTGCCGGATCGCTTTAGCGGTAGTCGTTATGGTACCGATTTTACCCTGACC

[0139] ATTAGTAGTCTGCAGCCGGAAGATATTGCCACCTATTATTGTCAGCAGTATAGTGAT

[0140] CCGCCGCTGACCTTTGGCGGTGGTACAAAAGTTGAAATTGGTGGTAGCGGCGGTAG

[0141] TGGTGGTAGTGATGATGATGATGACAAAGGTGGCAGCGGTGGTAGTTTTGTTAATA

[0142] AACAGTTCAACTACAAGGACCCGGTTAATGGCGTGGATATTGCCTATATTAAGATCC

[0143] CGAATGTGGGCCAGATGCAGCCGGTTAAAGCCTTTAAAATTCATAACAAGATCTGG

[0144] GTGATCCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGCGATCTGAATCCGCC

[0145] GCCTGAAGCCAAACAGGTGCCGGTGTCATATTATGATAGTACCTATCTGAGCACCG

[0146] ATAATGAAAAAGATAACTACCTGAAGGGCGTGACCAAACTGTTTGAACGCATTTAT

[0147] AGCACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTGCGCGGCATTCCGTTTTGG

[0148] GGTGGCTCAACCATTGATACCGAACTGAAAGTTATCGATACCAATTGCATCAACGT

[0149] GATTCAGCCGGATGGTAGTTATCGCAGTGAAGAACTGAATCTGGTTATTATTGGCCC

[0150] GAGCGCCGATATTATTCAGTTTGAATGTAAGAGCTTCGGCCATGAAGTGCTGAATCT

[0151] GACCCGTAATGGTTATGGCAGCACCCAGTATATTCGCTTTAGTCCGGATTTTACCTT

[0152] CGGTTTGAAGAAAGCCTGGAAGTTGATACCAATCCGCTGCTGGGTGCAGGTAAAT

[0153] TTGCAACCGATCCGGCCGTGACCCTTGGCTCACGAGTTAATTCATGCCGGTCATCGCC

[0154] TGTATGGCATTGCAATTAATCCGAATCGTGTTTTTAAGGTGAACACCAATGCATATT

[0155] ACGAGATGAGCGGTCTGGAAGTTAGCTTTGAAGAACTGCGTACCTTTGGTGGTCAT

[0156] GATGCCAAATTTATTGACAGTCTGCAGGAAAATGAGTTCCGCCTGTATTATTATAAC

[0157] AAGTTCAAGGACATCGCAAGCACCCTGAATAAAGCCAAAAGCATTGTGGGTACCAC

[0158] CGCAAGCCTGCAGTATATGAAAAATGTTTTCAAGGAGAAGTACCTGCTGAGTGAAG

[0159] ATACCAGCGGTAAATTTAGTGTTGATAAGCTGAAGTTCGACAAGCTGTATAAAATG

[0160] CTGACCGAAATTTACACCGAGGATAATTTTGTGAAGTTCTTCAAGGTGCTGAACCGT

[0161] AAAACCTATCTGAATTTTGACAAGGCAGGTGTTTAAGATCAACATCGTGCCGAAAGT

[0162] GAATTATACCATTTACGATGGCTTCAACCTGCGCAATACCAATCTGGCAGCCAATTT

[0163] TAATGGTCAGAATACCGAATCAACAACAATGAATTTCACCAAGCTGAAGAACTTCA

[0164] CCGGTCTGTTTGAATTTTACAAGCTGCTGTGCGTTCGTGGTATTATTACCAGTGGTG

[0165] GTAGCGATGATGATGATAAAGGCGGTAGTGCACTGAATGATCTGTGTATTAAAGTG

[0166] AACAACTGGGATCTGTTCTTCAGTCCGAGCGAAGATAATTTTACCAATGATCTGAAC

[0167] AAGGGCGAAGAAATTACCAGCGATACCAATATTGAGGCCGCAGAAGAAAATATTA

[0168] GCCTGGATCTGATTCAGCAGTATTATCTGACCTTTAACTTCGATAACGAGCCGGAAA

[0169] ATATCAGTATTGAAAACCTGAGTAGCGACATTATCGGTCAGCTGGAACTGATGCCG

[0170] AATATTGAACGCTTTCCGAATGGTAAAAAGTACGAACTGGATAAATACACCATGTT

[0171] CCATTACCTGCGCGCACAGGAATTTGAACATGGCAAAAGCCGCATTGCACTGACCA

[0172] ATAGTGTGAATGAAGCACTGCTGAATCCGAGCCGCGTGTATACCTTTTTTAGTAGTG

[0173] ATTACGTGAAGAAGGTGAACAAAGCCACCGAAGCAGCAATGTTTCTGGGCTGGGTT

[0174] GAACAGCTGGTTTATGATTTTACCGATGAAACCAGCGAAGTTAGCACCACCGATAA

[0175] AATTGCCGATATTACCATTATCATCCCGTATATTGGCCCGGCACTGAATATTGGCAA

[0176] TATGCTGTATAAGGACGACTTTGTGGGCGCCCTGATTTTTAGCGGCGCCGTTATTCT

[0177] GCTGGAATTTATTCCGGAAATCGCCATTCCGGTGCTGGGTACCTTTGCACTGGTTAG

[0178] TTATATTGCCAATAAGGTGCTGACCGTTCAGACCATTGATAATGCACTGAGCAAACG

[0179] CAATGAAAAATGGGATGAAGTTTACAAGTACATCGTGACCAATTGGCTGGCCAAAG

[0180] TTAATACCCAGATTGATCTGATTCGCAAAAAGATGAAGGAGGCACTGGAAAATCAG

[0181] GCCGAAGCAACCAAAGCAATTATTACCAGTACAACAGTACACCGAAAGA

[0182] AAAAAACAACATCAACTTCAACATCGACGACCTGAGCAGCAAACTGAATGAAAGT

[0183] ATTAACAGGCCATGATCAACATCAACAAGTTTCTGAATCAGTGCAGTGTGAGTTAT

[0184] CTGATGAATAGCATGATCCCGTATGGTGTTAAACGTCTGGAAGATTTTGACGCCAGGC

[0185] CTGAAAGATGCCCTGCTGAAATATATTTACGACAATCGTGGCACCCTGATTGGTCAG

[0186] GTGGATCGTCTGAAAGATAAAGTTAATAACACCCTGAGCACCGACATTCCGTTTCA

[0187] GCTGAGCAAATATGTTGATAATCAGCGCCTGCTGAGTACCTTTACCGAATATTAA

[0188] GAACATCATCAACACCAGCATCCTGAATCTGCGTTATGAAAGCAATCATCTGATCG

[0189] ATCTGAGCCGTTATGCCAGCAAAATTAATATTGGTAGCAAGGTTAACTTCGACCCGA

[0190] TTGATAAAAACCAGATTCAGCTGTTTAACCTGGAAAGCAGTAAAATTGAGGTGATT

[0191] CTGAAGAACGCAATCGTTTATAACAGCATGTACGAAAACTTCAGCACCAGTTTTTGG

[0192] ATTCGCATTCCGAAATATTTCAACAGCATCAGTCTGAATAACGAGTATACCATTATC

[0193] AACTGCATGGAAAACAACAGTGGCTGGAAAGTTAGCCTGAATTATGGCGAAATTAT

[0194] CTGGACCCTGCAGGATACCCAGGAAATTAAACAGCGTGTGGTTTTTAAGTACAGCC

[0195] AGATGATTAACATCAGCGATTATATCAACCGCTGGATTTTTGTGACCATTACAAATA

[0196] ACCGCCTGAATAATAGCAAGATCTACATTAACGGCCGCCTGATTGATCAGAAACCG

[0197] ATTAGTAATCTGGGTAATATCCACGCAAGCAATAATATTATGTTCAAGCTGGACGGT

[0198] TGCCGTGATACCCATCGTTATATTTGGATTAAGTACTTCAACCTGTTCGACAAAGAG

[0199] CTGAATGAAAAAGAAATCAAGGACCTGTACGACAATCAGAGTAATAGCGGTATTCT

[0200] GAAAGACTTTTGGGGTGATTATCTGCAGTATGATAAGCCGTATTACATGCTGAATCT

[0201] GTATGATCCGAACAAATACGTTGACGTTAACAATGTGGGTATCCGTGGTTATATGTA

[0202] TCTGAAAGGTCCGCGCGGTAGCGTTATGACCACCAATATTTATCTGAACAGCAGCCT

[0203] GTATCGCGGTACCAAATTTATTATTAAGAAGTACGCAAGCGGTAACAAGGATAATA

[0204] TTGTTCGTAACAACGACCGCGTTTATATTAATGTTGTGGTTAAGAACAAGGAGTACC

[0205] GCCTGGCCACCAATGCAAGCCAGGCTGGTGTTGAAAAAATTCTGAGTGCACTGGAA

[0206] ATCCCGGATGTGGGCAATCTGAGTCAGGTTGTTGTTATGAAAAGCAAAAACGACCA

[0207] GGGTATTACCAATAAATGCAAAATGAACCTGCAGGATAACAACGGCAATGATATTG

[0208] GCTTTATCGGCTTTCATCAGTTTAACAATATCGCAAAGCTGGTGGCCAGTAATTGGT

[0209] ATAATCGCCAGATTGAACGTAGCAGTCGTACCCTGGGTTGCAGTTGGGAATTTATTCCTGTGGATGATGGTTGGGGTGAACGCCCGCTGTGA。

[0210] Example 1: Sequence and Expression Plasmid Construction Method

[0211] The amino acid sequence of the botulinum toxin type A protein used in the present invention is shown in SEQ ID NO: 1, wherein 1-435 is the light chain of the botulinum toxin type A protein, 438-443 is the enterokinase recognition cleavage site, and 446-1294 is the heavy chain of the botulinum toxin type A protein. The coding DNA sequence is designed according to the codon preference of Escherichia coli as shown in SEQ ID NO: 2, which is synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. PCR amplification is performed using the synthesized SEQ ID NO: 2 sequence as a template, and a coding DNA sequence of the enterokinase recognition sequence-DDDDK is added to the upstream primer. The PCR amplification products are cloned into expression vectors with different affinity tags through seamless connection, and Escherichia coli is transformed for expression and purification.

[0212] Example 2: Expression and purification of botulinum toxin using histidine or MBP as a tag

[0213] According to the method of Example 1, the sequence of SEQ ID NO:2 was cloned into the pET14b vector, transformed into Escherichia coli BL21 (DE3), and positive monoclonal bacteria were selected. The monoclonal bacteria were inoculated into a triangular flask containing 10ml LB / Amp medium and cultured overnight at 37°C and 150rpm. On the second day, all the overnight cultures were inoculated into a triangular flask containing 1L LB / Amp medium, cultured for 3h, and IPTG was added to a final concentration of 0.2mM for induction. At the same time, the culture temperature was reduced to 20°C. After 20h of induction culture, the bacteria were collected by centrifugation and ultrasonically disrupted. The supernatant after disruption was purified with 5ml Ni Bestarose FF filler (Bogelon Biotechnology Co., Ltd.), and after loading, it was rinsed with 20mM PB, 0.5M NaCl, and 10mM imidazole at pH7.0, and then eluted with 20mM PB, 0.5M NaCl, and 0.5M imidazole at pH7.0. The eluate was detected by SDS-PAGE.

[0214] According to the method of Example 1, the sequence of SEQ ID NO:2 was cloned into the pMAL-C6T vector, transformed into Escherichia coli BL21 (DE3), and positive monoclonal bacteria were selected. The monoclonal bacteria were inoculated into a triangular flask containing 10ml LB / Amp medium and cultured overnight at 37°C and 150rpm. On the second day, all the overnight cultures were inoculated into a triangular flask containing 1L LB / Amp medium, cultured for 3h, and IPTG was added to a final concentration of 0.2mM for induction. At the same time, the culture temperature was reduced to 20°C. After 20h of induction culture, the bacteria were collected by centrifugation and ultrasonically disrupted. The supernatant after disruption was purified with 5ml MBP Sepharose HP filler (Tian Di Renhe Biotechnology Co., Ltd.), and after loading, it was rinsed with 20mM Tris-HCl and 0.2M NaCl at pH7.0, and then eluted with 20mM Tris-HCl, 0.2M NaCl, and 10mM maltose at pH7.0. The eluate was detected by SDS-PAGE.

[0215] SDS-PAGE results are as follows Figure 1 As shown ( Figure 1 Middle: Lanes 1 and 5: protein molecular weight marker; Lane 2: purification loading with histidine as tag; Lane 3: purification flow-through with histidine as tag; Lane 4: purification elution with histidine as tag; Lane 6: purification loading with MBP as tag; Lane 7: purification flow-through with MBP as tag; Lane 8: purification elution with MBP as tag). Botulinum toxin protein was expressed by fusion with histidine or MBP as tag. Through low temperature induction, the fusion protein was expressed in soluble form and existed in the supernatant of bacterial cell fragmentation. After one-step affinity chromatography, the target protein was purified, but some impurity protein bands were still visible on the electrophoresis, and the purity of the target protein needed to be further improved. At the same time, the eluent with histidine as tag contained 0.5M imidazole, and enterokinase was inactive under this condition. The eluent could not be directly cleaved by enzyme, and ultrafiltration or chromatography was required to remove imidazole. Although the maltose in the eluate of the MBP tag has no effect on the activity of enterokinase, it still needs to be removed by ultrafiltration or chromatography after enzyme cleavage. Otherwise, the free tagged protein produced by enzyme cleavage cannot be removed by affinity using the MBP Sepharose HP filler.

[0216] Example 2: Expression of light chain variable region sequence and Protein L purification and design of light chain variable region-botulinum toxin fusion protein sequence

[0217] The sequence of the variable region of the antibody light chain used in the present invention is shown in SEQ ID NO:3, and the designed encoding DNA sequence is shown in SEQ ID NO:4, which is synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. The sequence of SEQ ID NO:4 was cloned into the pET22b vector through the Nco I / BamH I site and transformed into Escherichia coli BL21 (DE3). Positive monoclonal bacteria were selected and inoculated into a conical flask containing 1L LB / Amp medium, cultured at 37°C and 150rpm until OD600 = 0.8, IPTG was added to a final concentration of 0.2mM, cooled to 30°C, and continued to be cultured for 15h. The culture supernatant was collected by centrifugation, and the supernatant was purified with 5ml ProteinL filler (purchased from Boglon Biotechnology Co., Ltd.). After loading, it was eluted with 0.1M citric acid at pH 3.5, monitored and collected by UV280, and the eluate was adjusted to pH 7.0 with 1M Tris. The eluted sample was subjected to SDS-PAGE detection, and the results were as follows Figure 3 ( Figure 3 Middle: Lane 1: culture supernatant before induction of antibody light chain variable region expression bacteria; Lane 2: culture supernatant after induction of antibody light chain variable region expression bacteria; Lane 3: flow-through of culture supernatant after induction of antibody light chain variable region expression bacteria after Protein L affinity purification; Lane 4: elution of culture supernatant after induction of antibody light chain variable region expression bacteria after Protein L affinity purification; Lane 5: As shown by protein molecular weight Marker, the expressed light chain variable region is enriched and purified with a purity higher than 95%.

[0218] The verified light chain variable region sequence was used as a tag to splice with the SEQ ID NO:1 sequence, and a new protein molecular structure was obtained by adding a flexible peptide sequence and an enterokinase cleavage site sequence. Figure 2 As shown, the fusion protein is sequentially as follows from N-terminus to C-terminus: light chain variable region-flexible peptide-enterokinase site-flexible peptide-botulinum toxin light chain-flexible peptide-enterokinase-flexible peptide-botulinum toxin heavy chain. Its protein and nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0219] Example 3: Expression of light chain variable region-botulinum toxin fusion protein

[0220] The SEQ ID NO:6 sequence was cloned into the pET30a vector through the Nde I / Xho I site and transformed into Escherichia coli BL21 (DE3). The positive monoclonal bacteria were selected and sequenced for verification. The monoclonal bacteria were inoculated into a flask containing 10 ml of LB / Kana medium and cultured overnight at 37°C and 150 rpm. The next day, all the overnight cultures were inoculated into a flask containing 1 L of LB / Amp medium, cultured for 3 hours, and IPTG was added to a final concentration of 0.2 mM for induction, and the culture temperature was reduced to 20°C. After 20 hours of induction culture, the bacteria were collected by centrifugation.

[0221] Example 4: Purification of light chain variable region-botulinum toxin fusion protein

[0222] Resuspend the bacteria at a ratio of 10% with a buffer solution (10mM PB, 1mM EDTA, pH7.2), place on ice, and disrupt the bacteria with ultrasound. Centrifuge at 10000rpm for 10min and collect the supernatant. Take 10ml of ProteinL filler, balance it with an equilibrium buffer (10mMPB, 150mM NaCl, pH7.4), load the supernatant after the bacteria are disrupted, rinse with the equilibrium buffer until UV280 is stable, elute with elution buffer (0.1M citric acid, pH3.5), and immediately adjust to pH7.0 with 1M Tris after elution. The eluted sample was detected by SDS-PAGE. The expression and purification results of the light chain variable region-botulinum toxin fusion protein are shown in Figure 2. Figure 4 As shown, the fusion protein was specifically affinity purified, and the impurity protein was effectively removed. The purity of the fusion protein was higher than 95% through band grayscale scanning and SEC-HPLC analysis, which was significantly better than the histidine tag and MBP tag in Example 1.

[0223] Example 5: Activation of light chain variable region-botulinum toxin fusion protein

[0224] The fusion protein was adjusted to a protein concentration of 0.2 mg / ml and pH 8.0, and enterokinase (Yaxin Biotechnology Co., Ltd.) was added at a ratio of 0.2 U per 50 ug of fusion protein. After mixing, it was placed at 4°C for enzyme digestion for 48 h. The enzyme digestion results were detected by reducing and non-reducing SDS-PAGE. Figure 4 As shown, on non-reducing SDS-PAGE, the light chain variable region fragment used as a tag was removed after enzyme cleavage, and the molecular weight of the fusion protein was reduced. On reducing SDS-PAGE, the interchain disulfide bonds between the heavy and light chains of the recombinant botulinum toxin protein were opened, and separate light and heavy chain protein bands were visible, as well as a small amount of intact protein that was not cleaved and the product of partial enzyme cleavage: light chain variable region-flexible peptide-enterokinase site-flexible peptide-botulinum toxin light chain-flexible peptide.

[0225] Example 6: Removal and purification of light chain variable region-botulinum toxin fusion protein tag

[0226] The product after enzyme digestion was directly purified with 1 ml of Protein L filler, and the flow-through was collected after loading. The protein bound to the filler was eluted with elution buffer (0.1 M citric acid, pH 3.5), and both the flow-through and the eluate were subjected to SDS-PAGE detection. Figure 4 As shown ( Figure 4 Middle: Lanes 1 and 7: protein molecular weight marker; Lane 2: protein L purified loading; Lane 3: protein L purified flow-through; Lane 4: fusion protein obtained by protein L purification (non-reduced): light chain variable region-flexible peptide-enterokinase site-flexible peptide-botulinum toxin light chain-flexible peptide-enterokinase site-flexible peptide-botulinum toxin heavy chain; Lane 5: fusion protein after enterokinase digestion (non-reduced); Lane 6: flow-through purified by protein L after digestion (non-reduced); Lane 8: fusion protein after enterokinase digestion (reduced); Lane 9: flow-through purified by protein L after digestion (reduced); Lane 10: elution purified by protein L after digestion (reduced), the intact protein with the tag, some digestion products and free tags are re-bound to the filler, the flow-through contains only the target botulinum toxin protein, and the light chain and heavy chain are connected by disulfide bonds.

[0227] Example 7: Activity assay

[0228] The flow-through solution of Example 6 was diluted with physiological saline and injected into 10 26-30 day old female Kunming mice (SPF grade), with 0.5 ml injected intraperitoneally into each mouse. The mice were continuously observed after injection. By the fourth day, a total of 6 mice died, and the surviving mice all had symptoms such as ruffled hair, listlessness, and rapid breathing.

[0229] In summary, the present invention provides a recombinant botulinum toxin and a method for preparing the same. The tag protein used in the present invention is of moderate size and simple structure, and can be affinity purified with ProteinL filler, with high yield and good specificity. In particular, affinity purification elution can be achieved only by low pH, and enzyme cleavage can be performed after the eluent is adjusted to an appropriate pH. In addition, there is no need for ultrafiltration to remove the eluent and other operations, and the enzyme cleavage product can be affinity purified again to efficiently remove the tag protein and related impurities, which greatly simplifies the production steps and costs of botulinum toxin and makes up for the deficiencies of the prior art.

[0230] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0231] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A recombinant botulinum toxin, characterized in that: The recombinant botulinum toxin includes an antibody light chain variable region as a tag protein, and the protein sequence of the antibody light chain variable region is shown in SEQ ID NO:

3.

2. The recombinant botulinum toxin according to claim 1, characterized in that The DNA sequence encoding the antibody light chain variable region is shown in SEQ ID NO:

4.

3. The recombinant botulinum toxin according to claim 1, characterized in that The botulinum toxin includes botulinum toxin type A, and the amino acid sequence of the botulinum toxin type A is shown in SEQ ID NO:

1.

4. The recombinant botulinum toxin according to claim 3, characterized in that The DNA sequence encoding the botulinum toxin type A is shown in SEQ ID NO:

2.

5. The recombinant botulinum toxin according to claim 3, characterized in that As shown in SEQ ID NO: 1, in the amino acid sequence, 1-435 is the light chain of the type A botulinum toxin, 438-443 is the enterokinase recognition cleavage site, and 446-1294 is the heavy chain of the type A botulinum toxin.

6. The recombinant botulinum toxin according to claim 1, characterized in that The antibody light chain variable region-botulinum toxin fusion protein sequence is shown in SEQ ID NO:

5.

7. The recombinant botulinum toxin according to claim 1, characterized in that The nucleotide sequence of the antibody light chain variable region-botulinum toxin fusion protein is shown in SEQ ID NO:

6.

8. A method for preparing the recombinant botulinum toxin according to any one of claims 1 to 7, characterized in that: The preparation method comprises the following steps: Using the variable region of the antibody light chain as a tag protein, fusion expression is used to prepare recombinant botulinum toxin; The recombinant botulinum toxin is purified using Protein L filler.

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