Recombinant genetic engineering bacteria of botulinum toxin type A and its preparation and application

Abstract

The present invention provides a recombinant genetically engineered bacterium of type A botulinum toxin and its preparation and application, belonging to the field of genetic engineering, the recombinant genetically engineered bacterium includes a pET‑28a vector, the pET‑28a vector includes a gene fragment 1 expressing a GST tag, a gene fragment 2 expressing a thrombin recognition site, and a gene fragment 3 expressing BoNT / A; the amino acid sequence of the GST is SEQ ID NO.1; the amino acid sequence of the thrombin recognition site is SEQ ID NO.2; the amino acid sequence of BoNT / A is SEQ ID NO.3. The recombinant genetically engineered bacterium provided by the present invention can produce type A botulinum toxin quickly and in large quantities. The present invention also optimizes the culture method of the recombinant genetically engineered bacterium, and increases the yield of type A botulinum toxin by adding an inducer. The present invention also optimizes the purification process of type A botulinum toxin, and prepares high-purity type A botulinum toxin.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and in particular relates to a recombinant genetic engineering bacterium of type A botulinum toxin and its preparation and application. Background Art

[0002] Botulinum toxin is a bacterial exotoxin produced by Clostridium botulinum during its growth and reproduction. According to the different antigenicity of botulinum toxin, it can be divided into eight types: A, B, C1, C2, D, E, F, and G. The molecular weight of these toxins is around 150kD. At present, the main types that have been developed and applied in clinical practice are A and B.

[0003] Botulinum toxin type A (BoNT / A) is a polypeptide chain composed of a heavy chain and a light chain bound by a disulfide bond, which can be cleaved by proteases. Botulinum toxin type A blocks neuromuscular conduction and causes flaccid paralysis by cleaving proteins necessary for the release of acetylcholine. After injection, the toxin binds to the receptor sites of cholinergic nerve endings, and the heavy chain of the toxin peptide chain binds to the unmyelinated area of ​​the nerve endings. The toxin itself enters the nerve membrane, and acetylcholine is blocked in the cytoplasm by the light chain and cannot pass through the neuromuscular junction. The nerve no longer transmits the medium, and the muscle is paralyzed, thereby reducing muscle tension and relieving muscle spasms. Botulinum toxin type A is often used in the plastic surgery industry in the form of injections. It can act on muscle tissue and is often used to solve masseter hypertrophy. It can also be used for anti-wrinkle and other purposes.

[0004] The anti-wrinkle mechanism of botulinum toxin type A is to achieve the effect of treating wrinkles by blocking neuromuscular conduction and inhibiting muscle contraction. Affected by the amount of injection, weakness and relaxation of the target muscles can occur as early as 24 hours after injection, usually 48-72 hours after injection of the toxin. The peak effect is usually after 7-14 days and lasts for about 3-6 months.

[0005] Existing BoNT / A methods are complicated, have a long preparation process, contain a large amount of non-BoNT / A components, and have low purity.

[0006] Chinese patent application CN202210572145.3 discloses a method for preparing recombinant botulinum toxin type A. The method comprises: preparing by a gene recombination method, constructing genetically engineered bacteria that express light chain proteins and heavy chain proteins respectively; subjecting the light chain proteins to a first denaturation treatment to obtain a first denatured product; subjecting the heavy chain proteins to a second denaturation treatment to obtain a second denatured product; mixing the first denatured product and the second denatured product in proportion, performing renaturation and assembly treatment, and obtaining the botulinum toxin type A. However, this method involves problems such as protein denaturation and renaturation, and the yield is often not high.

[0007] Chinese patent application CN202211539717.4 discloses a recombinant botulinum neurotoxin and its preparation method and application. The recombinant botulinum neurotoxin of the invention inserts an exogenous protease recognition site to effectively improve the cleavage efficiency of the single-chain polypeptide, obtains a double-chain BoNT / A with high biological activity, and quantitatively and proportionally expresses the single-chain recombinant botulinum neurotoxin and the exogenous protease in Escherichia coli. The exogenous protease can cleave the single-chain recombinant botulinum neurotoxin in Escherichia coli to produce an active double-chain botulinum neurotoxin, thereby realizing one-step production using Escherichia coli. However, the impact of this method on the purification of later products is difficult to eliminate.

[0008] In order to achieve large-scale and high-quality production of botulinum toxin type A, it is necessary to continuously explore the construction methods of recombinant genetically engineered bacteria of botulinum toxin type A and the purification methods of subsequent products. Summary of the invention

[0009] In order to solve the above problems, the present invention constructs a recombinant genetically engineered bacterium expressing botulinum toxin type A through genetic engineering technology, so as to achieve the purpose of rapid and high-quality production of botulinum toxin type A.

[0010] In one aspect, the present invention provides a recombinant genetically engineered bacterium that expresses botulinum toxin type A.

[0011] The recombinant genetically engineered bacteria include a pET-28a vector, and the pET-28a vector includes a gene fragment 1 expressing a GST tag, a gene fragment 2 expressing a thrombin recognition site, and a gene fragment 3 expressing BoNT / A; the amino acid sequence of the GST is SEQ ID NO.1; the amino acid sequence of the thrombin recognition site is SEQ ID NO.2; and the amino acid sequence of the BoNT / A is SEQ ID NO.3.

[0012] SEQ ID NO.1:

[0013] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIA YSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD;

[0014] SEQ ID NO.2: LVPRGS;

[0015] SEQ ID NO.3:

[0016] PFVNKQFNYKDPVNGVDIAYIKIPNVGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYK LLCVRGIITSKTKS LVPRGSKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL*。

[0017] The light chain and heavy chain connecting region of the BoNT / A (SEQ ID NO.3) includes another thrombin recognition site with an amino acid sequence of SEQID NO.2. The expression gene of the thrombin recognition site on the BoNT / A is gene fragment 4, and the gene fragment 4 is located in the connecting region of the light chain fragment encoded and the heavy chain fragment encoded in gene fragment 3.

[0018] The thrombin recognition site expressed by the gene fragment 2 is at the N-terminus or C-terminus of the GST tag or BoNT / A, preferably at the C-terminus of the GST tag and / or the N-terminus of BoNT / ABoNT / A.

[0019] In some embodiments, the connection order of each gene fragment is:

[0020] Gene fragment 1-gene fragment 2-gene fragment 3.

[0021] Preferably, there are connecting peptides (GGGS) at both ends of the thrombin recognition site LVPRGS expressed by the gene fragment 2. n The connecting peptide exposes the tag and thrombin recognition site of the fusion protein, making it easier for thrombin to cut. Based on the above, the pET-28a vector can also include gene fragment 5 and gene fragment 6 expressing the connecting peptide. The connecting peptide expressed by the gene fragment 5 is located at the N-terminus of the thrombin recognition site LVPRGS expressed by the gene fragment 2, and the connecting peptide expressed by the gene fragment 6 is located at the C-terminus of the thrombin recognition site LVPRGS expressed by the gene fragment 2.

[0022] Specifically, the pET-28a vector includes:

[0023] Gene fragment 1-gene fragment 5-gene fragment 2-gene fragment 6-gene fragment 3.

[0024] The connecting peptides expressed by gene fragment 5 and gene fragment 6 may have different n values.

[0025] The connecting peptide (GGGS) n , where n is an integer ≥1.

[0026] The connecting peptides expressed by gene segment 5 and gene segment 6 are independently selected from the above ranges. For example, the n value of the connecting peptide expressed by gene segment 5 can be selected from any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.; the n value of the connecting peptide expressed by gene segment 6 can be selected from any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.

[0027] Preferably, n is an integer of 1-5, more preferably 1 or 2.

[0028] In some specific embodiments, the n value of the connecting peptide expressed by the gene segment 5 is 2; the n value of the connecting peptide expressed by the gene segment 6 is 1.

[0029] Based on codon degeneracy, on the basis of the above technical solutions, those skilled in the art can design a variety of gene sequences for constructing the pET-28a vector.

[0030] In some exemplary embodiments, the present invention provides sequences of specific gene fragments, such as:

[0031] The gene fragment 1 expressing the GST tag comprises the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having more than 80% identity with SEQ ID NO.4 and expressing the same amino acids;

[0032] SEQ ID NO.4:

[0033] ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCTTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGAATGAAGTGATAAATGGCGAAACAAAAAGTTTGAA TTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGC TTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATA TTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATT

[0034] GATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGC AAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGAT;

[0035] The gene fragment 2 or gene fragment 4 expressing the thrombin recognition site may be a nucleotide sequence as shown in any one of SEQ ID NOs. 5-6 or a nucleotide sequence having more than 80% identity with the nucleotide sequence as shown in any one of SEQ ID NOs. 5-6 and expressing the same amino acid;

[0036] SEQ ID NO.5: CTGGTTCCGCGTGGATCC;

[0037] SEQ ID NO. 6: CTGGTTCCGCGTGGTAGC.

[0038] The gene fragment 3 expressing BoNT / A may be a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having 80% or more identity with SEQ ID NO.7 and expressing the same amino acid; SEQ ID NO.7 contains SEQ ID NO.6. In some cases, a person skilled in the art may also try to replace SEQ ID NO.6 in SEQ ID NO.7 with SEQ ID NO.5;

[0039] SEQ ID NO.7:

[0040] CCGTTTGTGAACAAACAGTTCAACTATAAAGATCCGGTGAACGGTGTTGATATCGCCTATATCAAAATTCCGAATGTTGGTCAGATGCAGCCGGTTAAAGCCTTTAAAATCCATAACAAAATTTGGGTGATTCCGGAACGTGATACCTTTACCAATCCGGAAGAAGGTGATCTGAATCCGCCTCCGGAAGCAAAACAGGTTCCGGTTAGCTATTATGATAGCACCTATCTGAGCACCGATAACGAGAAAGATAACTATCTGAAAGGTGTGACCAAACTGTTTGAACGCATTTATAGTACCGATCTGGGTCGTATGCTGCTGACCAGCATTGTTCGTGGTATTCCGTTTTGGGGTGGTAGCACCATTGATACCGAACTGAAAGTTATTGACACCAACTGCATTAATGTGATTCAGCCGGATGGTAGCTATCGTAGCGAAGAACTGAATCTGGTTATTATTGGTCCGAGCGCAGATATCATTCAGTTTGAATGTAAAAGCTTTGGCCACGAAGTTCTGAATCTGACCCGTAATGGTTATGGTAGTACCCAGTATATTCGTTTCAGTCCGGATTTTACCTTTGGCTTTGAAGAAAGCCTGGAAGTTGATACAAATCCGCTGTTAGGTGCAGGTAAATTTGCAACCGATCCGGCAGTTACCCTGGCACATGAACTGATTCATGCAGGTCATCGTCTGTATGGTATTGCCATTAATCCGAATCGTGTGTTCAAAGTGAATACCAACGCCTATTATGAAATGAGCGGTCTGGAAGTGAGTTTTGAAGAACTGCGTACCTTTGGTGGTCATGATGCCAAATTTATCGATAGCCTGCAAGAAAATGAATTTCGCCTGTACTACTATAACAAATTCAAGGATATTGCGAGCACCCTGAATAAAGCCAAAAGCATTGTTGGCACCACCGCAAGCCT

[0041] GCAGTATATGAAAAATGTGTTTAAAGAAAAATATCTGCTGAGCGAAGATAC

[0042] CAGCGGTAAATTTAGCGTTGACAAACTGAAATTCGATAAACTGTACAAGAT

[0043] GCTGACCGAGATTTATACCGAAGATAACTTCGTGAAGTTTTTCAAAGTGCT

[0044] GAACCGCAAAACCTACCTGAACTTTGATAAAGCCGTGTTCAAAATCAACAT

[0045] CGTGCCGAAAGTGAACTATACCATCTATGATGGTTTTAACCTGCGCAATACC

[0046] AATCTGGCAGCAAACTTTAATGGTCAGAACACCGAAATCAACAACATGAA

[0047] CTTTACCAAACTGAAGAACTTCACCGGTCTGTTCGAATTTTACAAACTGCT

[0048] GTGTGTTCGTGGCATTATTACCAGCAAAACCAAAAGCCTGGTTCCGCGTGG

[0049] TAGCAAAGCCCTGAATGATCTGTGTATTAAAGTGAACAATTGGGACCTGTT

[0050] TTTCAGCCCGAGTGAAGATAATTTCACCAACGATCTGAACAAAGGCGAAG

[0051] AAATTACCAGCGATACCAATATTGAAGCAGCCGAAGAAAACATTAGCCTGG

[0052] ATCTGATTCAGCAGTATTATCTGACCTTCAACTTCGATAATGAGCCGGAAAA

[0053] TATCAGCATTGAAAACCTGAGCAGCGATATTATTGGCCAGCTGGAACTGAT

[0054] GCCGAATATTGAACGTTTTCCGAACGGCAAAAAGTACGAGCTGGATAAATA

[0055] CACCATGTTCCATTATCTGCGTGCCCAAGAATTTGAACATGGTAAAAGCCGT

[0056] ATTGCACTGACCAATAGCGTTAATGAAGCACTGCTGAACCCGAGCCGTGTT

[0057] TATACCTTTTTCAGCAGCGATTATGTGAAGAAGGTTAACAAAGCAACCGAA

[0058] GCAGCAATGTTTTTAGGTTGGGTTGAACAGCTGGTGTATGATTTCACCGATG

[0059] AAACCAGCGAAGTTAGCACCACCGATAAAATTGCAGATATTACCATCATCAT

[0060] CCCGTATATCGGTCCGGCACTGAATATTGGCAATATGCTGTATAAAGACGAT

[0061] TTTGTGGGTGCCCTGATTTTTAGCGGTGCAGTTATTCTGCTGGAATTTATTCC

[0062] GGAAATTGCCATTCCGGTTCTGGGCACCTTTGCACTGGTGAGCTATATTGCA

[0063] AATAAAGTTCTGACCGTGCAGACCATCGATAATGCACTGAGCAAACGTAAC

[0064] GAAAAAATGGGATGAAGTTGTACAAGTATATCGTGACCAATTGGCTGGCAAAA

[0065] GTTAACACCCAGATTGATCTGATCCGTAAAAAGATGAAAGAAGCCCTGGAA

[0066] AATCAGGCAGAAGCAACCAAAGCCATTATCAACTATCAGTATAACCAGTAC

[0067] ACCGAGGAAGAGAAGAATAACATCAACTTCAACATCGATGATCTGAGCAG

[0068] CAAGCTGAATGAAAGCATCAACAAAGCCATGATTAACATTAACAAATTTCT

[0069] GAACCAGTGCAGCGTGAGCTATCTGATGAATAGCATGATTCCGTATGGTGTG

[0070] AAACGTCTGGAAGATTTTGATGCAAGCCTGAAAGATGCTCTGCTGAAATAT

[0071] ATCTATGATAATCGTGGCACCCTGATTGGTCAGGTTGATCGTCTGAAAGATA

[0072] AGGTGAATAATACCCTGAGTACCGATATTCCTTTTCAGCTGAGCAAATATGT

[0073] GGATAATCAGCGTCTGCTGTCAACCTTTACCGAATACATTAAGAACATCATC

[0074] AACACCAGCATTCTGAACCTGCGTTATGAAAGCAATCATCTGATCGATCTGT

[0075] CTCGCTATGCAAGCAAAATCAATATAGGCAGCAAGGTCAACTTTGACCCGA

[0076] TTGACAAAAATCAGATACAGCTGTTTAATCTGGAAAGCAGCAAAATCGAAG

[0077] TGATCCTGAAAAACGCCATTGTGTATAACAGCATGTATGAGAATTTCTCGAC

[0078] CAGCTTTTGGATTCGCATTCCGAAATACTTTAATAGCATCAGCCTGAACAAC

[0079] GAGTACACCATTATTAACTGCATGGAAAACAATAGCGGCTGGAAAGTTAGC

[0080] CTGAATTATGGTGAGATTATTTGGACCCTGCAGGATACCCAAGAAATCAAA

[0081] CAGCGTGTGGTTTTCAAATACAGCCAGATGATTAATATCAGCGACTATATCA

[0082] ACCGCTGGATTTTTGTGACCATTACCAATAATCGCCTGAATAACAGCAAGAT

[0083] CTATATTAACGGTCGCCTGATTGATCAGAAACCGATTAGTAATCTGGGCAAT

[0084] ATTCATGCGAGCAACAACATCATGTTTAAACTGGATGGTTGTCGTGATACCC

[0085] ATCGTTATATTTGGATCAAGTACTTCAACCTGTTCGATAAAGAGCTGAACGA

[0086] AAAAGAAATTAAAGATCTGTATGATAACCAGAGCAACAGCGGTATTCTGAA

[0087] AGACTTTTGGGGAGATTATCTGCAGTATGACAAACCGTATTATATGCTGAAC

[0088] CTGTACGACCCGAACAAATATGTTGATGTGAATAATGTGGGCATCCGTGGCT

[0089] ATATGTACCTGAAAGGTCCGCGTGGCAGCGTTATGACCACAAACATTTATCT

[0090] GAATAGCAGCCTGTATCGCGGTACGAAATTTATCATTAAAAAGTATGCCAGC

[0091] GGCAACAAAGATAACATTGTGCGTAATAATGATCGCGTGTATATCAATGTGG

[0092] TGGTGAAGAATAAAGAATATCGTCTGGCAACCAATGCAAGCCAGGCAGGC

[0093] GTTGAGAAAATTCTGAGCGCACTGGAAATTCCGGATGTGGGTAATCTGAGC

[0094] CAGGTTGTTGTGATGAAAAGCAAAAATGATCAGGGCATCACCAACAAGTG

[0095] CAAAATGAATCTGCAGGACAATAACGGCAACGATATTGGTTTTATTGGCTTC

[0096] CACCAGTTCAACAATATTGCGAAACTGGTTGCAAGCAATTGGTATAATCGTC

[0097] AGATTGAACGTAGCAGTCGTACCCTGGGTTGTAGCTGGGAATTTATCCCTGTGGATGATGGTTGGGGTGAACGTCCGCTGTAA.

[0098] The connecting peptide is (GGGS) 2 When the connecting peptide is GGGS, the nucleotide sequence of the gene fragment 5 or the gene fragment 6 may include SEQ ID NO.8 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.8 and expresses the same amino acid; when the connecting peptide is GGGS, the nucleotide sequence of the gene fragment 5 or the gene fragment 6 may include SEQ ID NO.9 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.9 and expresses the same amino acid.

[0099] SEQ ID NO.8: GGTGGTGGTTCAGGTGGTGGAAGC;

[0100] SEQ ID NO.9: GGTGGTGGTTCA.

[0101] In some more specific embodiments, the pET-28a vector includes any of the following:

[0102] (1) the nucleotide sequences shown in SEQ ID NO.4-SEQ ID NO.5-SEQ ID NO.7 or nucleotide sequences having 80% or more identity thereto and expressing the same amino acids;

[0103] (2) The nucleotide sequences shown in SEQ ID NO.4-SEQ ID NO.8-SEQ ID NO.5-SEQ ID NO.9-SEQ ID NO.7 or nucleotide sequences having 80% or more identity thereto and expressing the same amino acids.

[0104] Specifically, the pET-28a vector may also include a gene fragment 7 expressing MGSS and a gene fragment 8 expressing a histidine tag. The gene fragment 7 and the gene fragment 8 are sequentially connected and then connected to the gene fragment 1; the histidine tag is preferably 6His.

[0105] More specifically, the pET-28a vector includes any of the following:

[0106] (1) gene segment 7-gene segment 8-gene segment 1-gene segment 2-gene segment 3;

[0107] (2) Gene fragment 7-Gene fragment 8-Gene fragment 1-Gene fragment 5-Gene fragment 2-Gene fragment 6-Gene fragment 3.

[0108] As a preferred example, the nucleotide sequence of the gene fragment 7 can be SEQ ID NO.10: ATGGGTAGCAGCCA, or a nucleotide sequence that has more than 80% identity with SEQ ID NO.10 and expresses the same amino acid; the gene fragment 8 can be SEQ ID NO.11: CATCATCATCATCACCAT, or a nucleotide sequence that has more than 80% identity with SEQ ID NO.11 and expresses the same amino acid.

[0109] Further preferably, the pET-28a vector comprises any of the following:

[0110] (1) the nucleotide sequences shown in SEQ ID NO.10-SEQ ID NO.11-SEQ ID NO.4-SEQ ID NO.5-SEQ ID NO.7 or nucleotide sequences having 80% or more identity thereto and expressing the same amino acids;

[0111] (2) The nucleotide sequences shown in SEQ ID NO.10-SEQ ID NO.11-SEQ ID NO.4-SEQ ID NO.8-SEQ ID NO.5-SEQ ID NO.9-SEQ ID NO.7, or nucleotide sequences having 80% or more identity thereto and expressing the same amino acids.

[0112] Those skilled in the art can synthesize the target gene by conventional means or quickly obtain a large number of fragments by amplification, or any other disclosed or undisclosed method in the prior art to obtain the target gene of the present invention.

[0113] Regarding the insertion method of the above gene into the pET-28a vector, those skilled in the art can simply select a method based on the knowledge of the technical solution of the present invention, and the resulting technical solution is also within the protection scope of the present invention. Fig.14 As shown, those skilled in the art can select the insertion site thereon as needed, preferably a double-site insertion, and in some cases a single-site insertion may be a feasible solution.

[0114] As an example, the insertion site of the above gene on the pET-28a vector can be any one or two of EcoRI, NcoI, BstEⅡ, SphⅠ, NruⅠ, PasⅠ, PmeⅠ, BamHI or RsrⅡ.

[0115] Preferably, the insertion site of the above gene in the pET-28a vector may be EcoRI and / or NcoI, more preferably EcoRI / NcoI.

[0116] The recombinant genetically engineered bacteria is preferably Escherichia coli, and more preferably Escherichia coli BL21 or ShuffleT7K12 strains, which are strains disclosed in the art.

[0117] The E. coli BL21 lacks outer membrane alkyl protease because the cell line does not include ion protease. This genotype of E. coli can reduce the degradation of heterologous proteins expressed in the cell line and is usually used to induce toxic protein expression. Gene expression is regulated by adding inducers to the culture.

[0118] The Shuffle T7K12 strain is derived from the Escherichia coli K12 strain and is suitable for protein expression initiated by the T7 promoter. The disulfide bond isomerase DsbC constitutively expressed in the cell is not only conducive to the formation of correct disulfide bonds in the expressed protein, but also has the function of a molecular chaperone to help proteins without disulfide bonds to fold correctly. The T7 RNA polymerase gene is integrated into the lac operon region on the bacterial chromosome. The constitutively expressed lac repressor protein can reduce the background expression of the gene, which is conducive to the expression of toxic proteins. It has no λ prophage sequence and has the characteristics of resistance to T1 phage infection.

[0119] On the other hand, the present invention provides a method for constructing the aforementioned recombinant genetically engineered bacteria.

[0120] Specifically, the construction method includes inserting each gene fragment into the pET-28a vector in sequence.

[0121] The embodiment of "sequential insertion" can be to construct multiple expression vectors and insert the target fragments sequentially, or to obtain the full sequence containing all target genes sequentially by chemical synthesis and then insert it into the expression vector.

[0122] In yet another aspect, the present invention provides the use of the aforementioned recombinant genetically engineered bacteria in the preparation of botulinum toxin type A.

[0123] Specifically, botulinum toxin type A is expressed by recombinant genetically engineered bacteria.

[0124] In another aspect, the present invention provides a method for preparing botulinum toxin type A.

[0125] The preparation method includes using the aforementioned recombinant genetic engineering bacteria for expression.

[0126] The expression condition is that the recombinant genetic engineering bacteria are inoculated into a culture medium and cultured at 35-40°C.

[0127] Preferably, the culture medium types include but are not limited to LB medium, SOB medium, TB medium, mTB medium, and YT medium.

[0128] Preferably, the expression condition is that the recombinant genetically engineered bacteria are inoculated into a culture medium and cultured at 30-37°C.

[0129] The expression conditions may also selectively include the addition of an inducer.

[0130] The inducing agent may be added at a concentration of 0-1 mM, preferably 0.4-1 mM, and more preferably 0.4 mM.

[0131] The type of the inducer can be IPTG, lactose or L-arabinose.

[0132] When the inducer is lactose, the induction concentration is 0.1-0.5 mmol / L, and the induction temperature is 16-25°C.

[0133] Specifically, the preparation method may also include the purification of botulinum toxin type A, and the purification includes any one or more of centrifugation, homogenization, clarification, NTA affinity chromatography, hydrophobic chromatography, G25 fluid exchange, SP ion exchange, thrombin cleavage, GST affinity chromatography, and G200 molecular sieve.

[0134] The purification may be a combination of centrifugation, homogenization and clarification; the specific conditions of the purification may be adjusted by those skilled in the art according to routine procedures.

[0135] Preferably, the purification comprises: NTA affinity chromatography, hydrophobic chromatography, G25 fluid exchange and SP ion exchange.

[0136] Further preferably, the purification further comprises: any one or more of thrombin enzymatic cleavage, GST affinity chromatography, and G200 molecular sieve, that is, any one or more of the thrombin enzymatic cleavage, GST affinity chromatography, and G200 molecular sieve are combined with NTA affinity chromatography, hydrophobic chromatography, G25 fluid exchange, and SP ion exchange to complete the purification operation.

[0137] As for the specific reagents involved, those skilled in the art can choose them as needed. For example, the thrombin in the thrombin digestion can be selected from thrombin extracted from nature or prepared by genetic engineering, and can be from a variety of animal sources, such as: humans, cattle, pigs, rabbits, mice, sheep, dogs, etc.; the genetic engineering includes but is not limited to genetic engineering expression and genetic engineering modification.

[0138] The present invention also protects the botulinum toxin type A prepared according to the aforementioned preparation method and the product containing the botulinum toxin type A.

[0139] The product may be a medical cosmetic product or a medicine.

[0140] The types of products include but are not limited to injections and smear-type preparations.

[0141] The product may also include corresponding auxiliary materials commonly used in the art.

[0142] For example, when the product is an injection, those skilled in the art know that it may also include inactive ingredients allowed to be used in injections, such as buffer components, stabilizing components, dispersing components, excipient components, etc. More specifically, it may include: sodium chloride, polyvinyl pyrrolidone, gelatin, glycerol, polysorbate 80, human albumin, lactose dextran, sucrose, gelatin, etc.

[0143] The product may be in a solid, semi-solid or liquid state.

[0144] As described, the product may be a lyophilized powder, which may be dissolved in a solvent for further use before injection.

[0145] Other applicable specific product types known to those skilled in the art, which can be realized by conventional means according to the technical solution provided by the present invention, also fall within the protection scope of the present invention.

[0146] Beneficial effects of the present invention:

[0147] The recombinant genetically engineered bacteria expressing botulinum toxin type A provided by the present invention can produce botulinum toxin type A quickly and in large quantities. The present invention also optimizes the culture method of the recombinant genetically engineered bacteria and increases the yield of botulinum toxin type A by adding an inducer. The present invention further optimizes the botulinum toxin type A purification process and prepares high-purity botulinum toxin type A. The overall solution of the present invention has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0148] Figure 1 The electrophoresis diagram is the full-length amplification of different gene sequences.

[0149] Figure 2-Figure 4 The results of screening for high-expression strains containing vectors with sequence numbers 2-4; wherein, Figure 2 After the competent cells of the STK model were transformed into fusion protein plasmids containing three different tags, GST, DsbA-GST, and NusA-GST, the SDS-PAGE experimental results of the target protein expressed by the corresponding plasmids; Figure 3 The SDS-PAGE experimental results of the target protein expressed by the corresponding plasmids after the TE-type competent cells were transformed into fusion protein plasmids containing three different tags: GST, DsbA-GST, and NuSA-GST; Figure 4 The SDS-PAGE experimental results of the target protein expressed by the corresponding plasmids after the BL21 competent cells were transformed into fusion protein plasmids containing three different tags: GST, DsbA-GST, and NuSA-GST.

[0150] Figure 5-Figure 6 The expression results of target proteins under different cell gene combination conditions; among them, Figure 5 The results of the GST activity detection experiment of three types of cells combined with vectors containing three different tags; Figure 6 The figures are the results of Western Blot experiments of three types of cells and vectors containing three different tags.

[0151] Figure 7-Figure 9 The results show the effect of different inducer concentrations on the expression of target protein in BL21-pET-28a-GST-BONTA cell gene combination. Figure 7 The results of the GST activity detection experiment of the target protein under different inducer concentrations of the BL21GST combination; Figure 8 The results of Western Blot experiments of the target protein of BL21GST combination at different inducer concentrations; Fig. 9 for Figure 8 The gray value of the middle band was obtained by grayscale analysis using GelPro software.

[0152] Fig.10This is the result of SDS-PAGE experiment on the sample after PG200 molecular sieve chromatography containing the target protein after removal of the GST tag.

[0153] Fig.11 The results of SDS-PAGE experiment after the target protein was digested with thrombin.

[0154] Fig.12 These are the results of SDS-PAGE experiments after thrombin digestion of the target proteins in different purified samples.

[0155] Fig.13 The results of SDS-PAGE experiments after thrombin digestion of the target protein without GST tag.

[0156] Fig.14 This is the plasmid map of the pET-28a vector backbone.

[0157] Fig.15 This is the plasmid map of pET-28a-MGSS-6His-GST-GGGGSGGGS-thrombin-GGGS-BONTA.

[0158] Fig.16 This is the pET-28a-MGSS-6His-GST-thrombin-bonta plasmid map. DETAILED DESCRIPTION

[0159] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0160] In the present invention, when it comes to the expression of common proteins, those skilled in the art can select competent cells including but not limited to E. coli BL21 or E. coli BL21 (DE3) as needed; the E. coli BL21 is suitable for high-level expression of non-toxic proteins, and cannot be used for protein expression driven by T7 promoter (such as pET series); the E. coli BL21 (DE3) is a λDE3 lysogen with T7 RNA polymerase, which can be used to express the target gene controlled by T7 promoter, and can also express genes controlled by E. coli promoters lac, tac, trc and trp.

[0161] In the present invention, when it comes to the expression of toxic proteins, those skilled in the art can select competent cells including but not limited to E. coli BL21 (DE3) plysS, OverExpress C43 (DE3) or BL21Gold pLysS (DE3) as needed, wherein:

[0162] The E. coli BL21 (DE3) plysS has the advantages of BL21 (DE3), and PlysS contains a gene for expressing T7 lysozyme. T7 lysozyme can act on the peptidoglycan on the cell wall of E. coli to dissolve E. coli, and can reduce the background expression level of the target gene, but does not interfere with the expression of the target protein, so it is suitable for expressing toxic proteins.

[0163] The two strains of OverExpress C43 (DE3) are both derived from BL21 (DE3), and their advantage is that they can efficiently express toxic or hydrophobic proteins. OverExpressC43 (DE3) is derived from OverExpressC41 (DE3), and is obtained by screening OverExpressC41 (DE3) resistant strains to another different toxic protein. The OverExpressC43 (DE3) strain has a stronger ability to express toxic and hydrophobic proteins than OverExpressC41 (DE3), and can be used for protein expression of plasmids such as pET series, pGEX, and pMAL.

[0164] The BL21Gold pLysS (DE3) is used for high-level expression of proteins, especially when expressed simultaneously with CE6 phage, it can be more strictly regulated and can be used for the expression of toxic proteins; this bacterial solution can be suitable for expression in non-T7 RNA polymerase systems.

[0165] In the present invention, when it comes to the expression of proteins containing rare codons, those skilled in the art can select competent cells including but not limited to E. coli BL21 (DE3) Condon Plus or E. coli Rosetta (DE3) as needed; the E. coli BL21 (DE3) Condon Plus is used for prokaryotic expression, lacks Lon protease and OmpT protease, and supplements 4 rare codons (AGA, AUA, CCC, CUA) lacking in E. coli; improves the expression level of AT- or GC-rich eukaryotic genes in prokaryotic systems; the E. coli Rosetta (DE3) supplements tRNA corresponding to 6 rare codons (AUA, AGG, AGA, CUA, CCC, GGA) lacking in E. coli, thereby improving the expression level of exogenous genes, especially eukaryotic genes, in prokaryotic systems.

[0166] In the present invention, when it comes to the expression of active proteins containing disulfide bonds, those skilled in the art can select competent cells as needed, including but not limited to: E. coli BL21 Origami B (DE3), E. coli Origami2 (DE3), BL21 trxB (DE3) or AD494 (DE3); wherein:

[0167] E.coli BL21Origami B (DE3) is a lacZY gene mutant derived from BL21; Origami B series strains combine the advantages of BL21, Tuner and Origami strains. Disulfide bonds are generated in the cytoplasm, which helps the formation of active proteins containing disulfide bonds. They are also resistant to tetracycline and kanamycin.

[0168] E.coli Origami 2 (DE3) is derived from K-12 cells and contains mutations in both trxB and gor genes. This strain can more efficiently generate disulfide bonds in the cytoplasm, which helps the formation of active proteins containing disulfide bonds. Compared with the Origami series of bacteria, the Origami 2 series of competent cells are kanamycin-sensitive, which makes this strain suitable for most protein expression plasmids. It has tetracycline resistance and streptomycin resistance;

[0169] BL21trxB (DE3) is derived from the BL21 strain, which is conducive to the formation of disulfide bonds of the target protein in the cytoplasm and can effectively improve the correct folding and expression of disulfide-bonded proteins. The BL21trxB host bacteria has the same thioredoxin reductase mutant TrxB mutant as the AD494 strain. The bacteria are resistant to kanamycin, so this strain can only be used for the expression of ampicillin-resistant plasmids;

[0170] AD494(DE3) is derived from E. coli κ-12 strain, a TrxB mutant with kanamycin resistance, so this strain can only be used for the expression of ampicillin-resistant plasmids.

[0171] In the present invention, when it comes to the expression of active proteins containing rare codons / disulfide bond folding, those skilled in the art can select competent cells including but not limited to Rosetta-gami (DE3) pLysS, Rosetta-gami B (DE3) or Rosetta-gami 2 (DE3) as needed, wherein:

[0172] Rosetta-gami (DE3) pLysS is derived from the Origami series of host bacteria. The advantages of this series of strains include the original TrxB and Gor gene mutations of the Origami strains to improve the efficient expression of disulfide bond-containing correctly folded proteins, and the rare codons of eukaryotic cells contained in the Rosetta series of strains. It has the advantages of both Rosetta strains and Origami strains, and is resistant to four antibiotics: chloramphenicol, kanamycin, streptomycin and tetracycline.

[0173] Rosetta-gamiB (DE3) is derived from the Origami B series strains, which combines the advantages of the BL21 series, Rosetta series strains and Origami series strains, greatly enhancing the formation of intracellular disulfide bonds and the expression of eukaryotic cell proteins containing rare codons in E. coli;

[0174] Rosetta-gami 2 (DE3) is derived from the Origami 2 series of strains. The Origami 2 series of strains are kanamycin-sensitive K-12 cell strains that carry mutations in the TrxB and Gor genes to improve the efficient expression of correctly folded proteins containing disulfide bonds.

[0175] In the present invention, when it comes to the expression of prokaryotic proteins of the pMAL series plasmids of NEB, those skilled in the art can select competent cells including but not limited to TB1 as needed, which lacks the T7 RNA polymerase of BL21 (DE3) and cannot be used for the expression of the PET series plasmids.

[0176] In the present invention, when it comes to IPTG stable expression, those skilled in the art can select competent cells including but not limited to Tuner (DE3) as needed. Tuner (DE3) strain is a lacZY gene (galactoside permease gene) mutant of BL21 strain. This mutation causes IPTG to enter each cell of Escherichia coli in the system at a uniform speed, resulting in a more stringent and uniform concentration dependence. It can be used for protein expression of plasmids such as pET series, pGEX, and pMAL.

[0177] In the present invention, when soluble expression is involved, those skilled in the art can select competent cells including but not limited to ArcticExpress (DE3) RP as needed, which can solve the common protein insolubility obstacle in bacterial gene expression. These cells are derived from high-performance BL21-Gold competent cells, allowing heterologous proteins to be expressed efficiently and at high levels in Escherichia coli.

[0178] In the present invention, the information of some constructed vectors used in the specific embodiments is shown in Table 1:

[0179] Table 1

[0180]

[0181] For the numbering of some vectors shown in the present invention, those skilled in the art can recognize the connection form of the nucleotide sequences included therein according to general understanding, such as "pET-28a-MGSS-6His-NusA-GST-thrombin-bonta" means that the nucleotide sequences (expression sequences) represented by MGSS, 6His, NusA, GST, thrombin, and bonta are sequentially connected on the pET-28a vector. Among them, the tag protein and the fusion protein, such as the tag protein 6HIS (6His), the GST fusion protein DsbA-GST, NusA-GST, TrxA-GST, etc. and the connecting peptide (GGGS) n It can be at the C-terminus of the target gene BONTA.

[0182] In the present invention, unless otherwise specified, some abbreviations have meanings commonly known in the art, such as "6His" means containing 6 histidine tags.

[0183] In the present invention, "GST" refers to glutathione S-transferase, and when it is located in a vector, it refers to the expression sequence (nucleotide sequence) of the enzyme.

[0184] In the present invention, "thrombin" refers to thrombin, and refers to the expression sequence of the thrombin cleavage site on the vector.

[0185] When referring to the botulinum toxin type A of the present invention, it can be referred to in the art in various forms, including but not limited to bonta, BoNT / A, and BONTA, all of which are related to botulinum toxin type A. Those skilled in the art can determine whether it is a nucleotide sequence or an amino acid sequence based on the understanding of the context.

[0186] The long fragment in Table 1 refers to the long expression fragment inserted into the corresponding vector, excluding the restriction site, among which the sequence represented by MGSS is SEQ ID NO.10; the sequence represented by 6His is SEQ ID NO.11; the sequence represented by GST is SEQ ID NO.4; the sequence represented by thrombin is SEQ ID NO.5; the sequence represented by bonta is SEQ ID NO.7; the sequence represented by GGGSGGGS is SEQ ID NO.8; the sequence represented by GGGS is SEQ ID NO.9; the sequence represented by DsbA is SEQ ID NO.12; the sequence represented by NusA is SEQ ID NO.13; and the sequence represented by TrxA is SEQ ID NO.14.

[0187] SEQ ID NO.12:

[0188] CATATGAAAAAGATTTGGCTGGCGCTGGCTGGTTTAGTTTTAGCGTTTAGCGCATCGGCGGCGCAGTATGAAGATGGTAAACAGTACACTACCCTGGAAAAACCGGTAGCTGGCGCGCCGCAAGTGCTGGAGTTTTTCTCTTTCTTCTGCCCGCACTGCTATCAGTTTGAAGAAGTTCTGCATATTTCTGATAATGTGAAGAAAAAACTGCCGGAAGGCGTGAAGATGACTAAATACCACGTCAACTTCATGGGTGGTGACCTGGGCAAAGATCTGACTCAGGCATGGGCTGTGGCGATGGCGCTGGGCGTGGAAGACAAAGTGACTGTTCCGCTGTTTGAAGGCGTACAGAAAACCCAGACCATTCGTTCTGCTTCTGATATCCGCGATGTATTTATCAACGCAGGTATTAAAGGTGAAGAGTACGACGCGGCGTGGAACAGCTTCGTGGTGAAATCTCTGGTCGCTCAGCAGGAAAAAGCTGCAGCTGACGTGCAATTGCGTGGCGTTCCGGCGATGTTTGTTAACGGTAAATATCAGCTGAATCCGCAGGGTATGGATACCAGCAATATGGATGTTTTTGTTCAGCAGTATGCTGATACAGTGAAATACTTAAGCGAGAAAAAA;

[0189] SEQ ID NO.13:

[0190]

[0191] SEQ ID NO.14:

[0192] ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTT GCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCTAACTCGAG.

[0193] In Table 1, when synthesizing long fragments, the restriction site-related sequences are designed according to the corresponding vectors, and the specific restriction sites have been disclosed in the vector restriction section of the specific examples.

[0194] Example 1

[0195] 1. The whole gene synthesis of SEQ ID NO. 1-8 in Table 1 was commissioned to Beijing Bomed Gene Technology Co., Ltd. Some electrophoresis results are shown in Figure 1 As shown, where:

[0196] Lane 1 is the DNA ruler (Maker);

[0197] Lane 2 is pET-28a-MGSS-6His-GST-thrombin-bonta long sequence + restriction site;

[0198] Lane 3 is pET-28a-MGSS-6His-DsbA-GST-thrombin-bonta long sequence + restriction site;

[0199] Lane 4 is pET-28a-MGSS-6His-NusA-GST-thrombin-bonta long sequence + restriction site;

[0200] Lane 5 is pET-28a-MGSS-6His-GST-GGGSGGGS-thrombin-bonta long sequence + restriction site;

[0201] Lane 6 is pET-28a-MGSS-6His-GST-GGGSGGGS-thrombin-GGGS-bonta long sequence + restriction site;

[0202] Lane 7 is pRSF-MGSS-6His-GST-thrombin-bonta-DsbA long sequence + restriction site;

[0203] Lane 8 is pRSF-MGSS-6His-GST-thrombin-bonta-TrxA long sequence + restriction site.

[0204] 2. Vector Digestion

[0205] (1) pET-28a was digested with NcoI and EcoRI. The digestion system is shown in Table 5:

[0206] Table 5

[0207] Element volume pET-28a 1 μg EcoRI-HF 1μL NcoI-HF 1μL rCutmart 2μL <![CDATA[dd H 2 The]]> Make up to 20 μL

[0208] The digestion conditions were as follows: digestion at 37°C for 1 h, followed by heat treatment at 80°C for 20 min to obtain the digestion product.

[0209] (2) pBAD24 restriction enzyme digestion system:

[0210] Element volume pBAD24 1ug EcoRI 1μL HindIII 1μL rCutmart 2μL dd H2O Make up to 20 μL

[0211] Enzyme digestion was performed at 37°C for 1 h, followed by heat treatment at 65°C for 20 min.

[0212] (3) pRSF enzyme digestion system:

[0213] Element volume pRSF-Duet-1 1ug EcoRI-HF 1μL NcoI-HF 1μL rCutmart 2μL dd H2O Make up to 20 μL

[0214] Enzyme digestion was performed at 37°C for 1 h, followed by heat treatment at 80°C for 20 min.

[0215] The three linear vector digestion products obtained above were connected in the next step.

[0216] 3. Reorganization connection

[0217] See Table 6 for general connection system:

[0218] Table 6

[0219]

[0220] Ligation reaction conditions: 50°C, 30 min. The ligation products were the 14 vectors shown in Table 1.

[0221] 4. Conversion reaction

[0222] 10 μL of the ligation product was added to 100 μL of NEB10β competent cells (Beijing Bomed), ice bathed for 30 min, 42°C water bathed for 1 min 30 s, and ice bathed for 2 min. 500 μL of SOB culture medium was added for recovery (37°C, 200 rpm shaker for 1 h). 200-300 μL was spread on the Kan plate and cultured in a 37°C incubator overnight (15 h). The SOB medium formula is as follows: 2.0% Tryptone, 0.5% Yeast Extract, 10 mM Sodium chloride, 2.5 mM Potassium Chloride, 10 mM Magnesium chloride, 10 mM Magnesium Sulfate.

[0223] 5. The obtained vectors were used to transform positive clones of different competent cells. Competent cells include: STK, TE, BL21. Competent cells and molecular biology reagents were provided by Beijing Biomed Gene Technology Co., Ltd.

[0224] VI. Positive clone screening

[0225] (1) Detection of target protein expression

[0226] The cell lysis method is to directly add 1× Loading buffer to the bacterial cells to lyse them. In the sample names of the result graph, DsbA-GST is abbreviated as D-GST, NuSA-GST is abbreviated as N-GST, and the digital numbers are the numbers of the four strains with the highest OD in the selected test tube samples (Note: This number has no corresponding relationship with Table 1, but is an error in parallel test screening).

[0227] The positive clones screened were detected by electrophoresis after cell lysis.

[0228] The operation of SDS-PAGE gel electrophoresis is as follows:

[0229] Loading buffer:

[0230] ①4× loading buffer (non-reduced): weigh 3.03 g of tris(hydroxymethyl)aminomethane (TRIS), 20 mg of bromophenol blue, 8.0 g of sodium dodecyl sulfate (SDS), 40 mL of glycerol, and 40 mL of pure water. Stir with a glass rod and use an ultrasonic cell washer until completely dissolved. Adjust the pH to 6.8 with hydrochloric acid and make the volume to 100 mL.

[0231] ②4× loading buffer (reduced form): Weigh 3.03 g of tris(hydroxymethyl)aminomethane (TRIS), 20 mg of bromophenol blue, 8.0 g of sodium dodecyl sulfate (SDS), 6.17 g of dithiothreitol (DTT), 40 mL of glycerol, and 40 mL of pure water. Stir with a glass rod and use an ultrasonic cell washer until completely dissolved. Adjust the pH to 6.8 with hydrochloric acid and make the volume to 100 mL.

[0232] MOPS Running Buffer liquid:

[0233] ①MOPS Running Buffer liquid (20×): MOPS electrophoresis buffer (20×), a commercial reagent purchased from Nanjing GenScript Biotechnology Co., Ltd.

[0234] ②MOPS Running Buffer liquid (1×): MOPS electrophoresis buffer (1×). Take 50 ml of MOPS Running Buffer liquid (20×), add pure water to 1 L, and mix by inverting.

[0235] Sample preparation:

[0236] Liquid sample: Add the corresponding liquid at a ratio of 4× loading buffer (non-reduced) to protein sample = 1:3. After adding, use a pipette to suspend and mix or use a vortex shaker to vortex and mix. After the metal bath temperature reaches the set temperature (99°C), place the mixed protein sample in the metal bath and heat at 99°C for 5 minutes. After heating, centrifuge at 14000×g for 1 minute.

[0237] Solid samples: directly add 1× loading buffer (reduced form) to the solid sample. The added volume is related to the mass of the solid sample. Generally, the added volume is 200μL. Use a pipette to suspend and mix. After the metal bath temperature reaches the set temperature (99°C), place the mixed protein sample in the metal bath and heat at 99°C for 15 minutes for denaturation. After heating, centrifuge at 14000×g for 1 minute.

[0238] Sample loading: Except for the marker, the sample loading volume per well of 11-well gel is generally 20μL, and the sample loading volume per well of 15-well gel is generally 10μL. When loading the sample, do not suck up the precipitate to avoid affecting the results.

[0239] Electrophoresis: Turn on the power switch of the electrophoresis instrument, press the "Select" button to set the voltage to 140V and the current to 400mA respectively, and end the electrophoresis when the blue blot of bromophenol blue runs to the bottom of the gel (about 66 minutes).

[0240] The screening results of high expression strains containing vectors with sequence numbers 2-4 are shown in Figure 2-Figure 4 .

[0241] Figure 2 The results of SDS-PAGE experiments showing that the competent cells of the STK model were transformed into fusion protein plasmids containing three different tags, GST, DsbA-GST, and NuSA-GST, and the corresponding plasmids expressed the target protein. The molecular weight of the target protein containing the GST tag is about 180Kda, the molecular weight of the target protein containing the DsbA-GST tag is about 200Kda, and the molecular weight of the target protein containing the NuSA-GST tag is about 230Kda. Lanes 2-5 are STK-GST combinations, lanes 6-9 are STK-DsbA-GST combinations, and lanes 10-13 are STK-NuSA-GST combinations. It can be concluded from this figure that among the three combinations, the expression level of the target protein corresponding to the STK-GST combination is the highest, the STK-NuSA-GST combination is the second highest, and the STK-DsbA-GST combination is the lowest.

[0242] Figure 3 The SDS-PAGE experimental results of the target protein expressed by the corresponding plasmids after the competent cells of the TE model were transformed into fusion protein plasmids containing three different tags, GST, DsbA-GST, and NuSA-GST. The molecular weight of the target protein containing the GST tag is about 180Kda, the molecular weight of the target protein containing the DsbA-GST tag is about 200Kda, and the molecular weight of the target protein containing the NuSA-GST tag is about 230Kda. Among them, lanes 2, 4-6 are TE-GST combinations, lanes 7-10 are TE-DsbA-GST combinations, and lanes 11-14 are TE-NuSA-GST combinations. It can be concluded from this figure that among the three combinations, the expression level of the target protein corresponding to the TE-DsbA-GST combination is higher, the TE-GST combination is the second highest, and the TE-NuSA-GST combination is the lowest.

[0243] Figure 4The SDS-PAGE experimental results of the target protein expressed by the corresponding plasmids after the competent cells of the BL21 model were transformed into fusion protein plasmids containing three different tags, GST, DsbA-GST, and NuSA-GST, respectively. The molecular weight of the target protein containing the GST tag is about 180Kda, the molecular weight of the target protein containing the DsbA-GST tag is about 200Kda, and the molecular weight of the target protein containing the NuSA-GST tag is about 230Kda. Among them, lanes 1-2, 4-5 are BL21GST combinations, lanes 6-9 are BL21DsbA-GST combinations, and lanes 10-13 are BL21NuSA-GST combinations. It can be concluded from this figure that among the three combinations, the expression level of the target protein corresponding to the BL21GST combination is higher, the BL21NuSA-GST combination is the second highest, and the BL21DsbA-GST combination is the lowest.

[0244] The other vectors in Table 1 were verified by referring to the above method, and the fusion protein containing GST tag was obtained by grayscale analysis:

[0245] BL21-pET-28a-MGSS-6His-GST-GGGGSGGGS-thrombin-GGGS-BONTA (corresponding to vector sequence number 7, plasmid map see Fig.15 );

[0246] BL21-pET-28a-MGSS-6His-GST-thrombin-bonta (corresponding to vector number 2, plasmid map see Fig.16 );

[0247] Has the highest expression level.

[0248] (2) Target protein activity detection - GST activity detection experiment

[0249] In order to further confirm that the expressed target protein is active, GST activity analysis was used to screen for genotype and cell type combinations with high activity and soluble expression. The sample for the GST activity detection experiment was bacterial lysis buffer, and the lysis method was to lyse the bacteria using IP lysis buffer, with a formula of 50mM Tris, 200mM NaCl, 2% Triton, and 0.1% SDS.

[0250] The GST activity detection experiment is introduced as follows: Glutathione S-transferase (GST) catalyzes the combination of reduced glutathione (GSH) and CDNB (1-Chlom-2,4-Dinitrobenzene, cDNB) to generate GS-DNB. The product has a characteristic absorption peak at 340nm. The change in the absorbance value of the product can characterize the rate of this enzymatic reaction. In a certain reaction system (25℃, pH6.0), the concentration of substrates GSH and CDNB is much greater than the concentration of enzyme GST, so the rate of the enzymatic reaction is proportional to the enzyme concentration.

[0251] GST is also a commonly used tag protein in the protein purification process. The fusion protein formed by the GST tag protein and the target protein also has the enzymatic activity of GST. Therefore, the rate of the enzymatic reaction also characterizes the concentration of the target protein to a certain extent.

[0252] During the detection process, 20 μL 1M KH 2 PO 4 (pH 6.5), 2μL 100mM CDNB, 2μL 100mM GSH, 20μL sample, fill to 200μL with water, build the reaction system, quickly mix and measure the absorbance at 340nm wavelength, measure once every 1min, total time 5min (measure 6 times). The result should be well linear, so after linear fitting of the data, its slope is the reaction rate value.

[0253] (3) Target protein activity detection-Western Blot

[0254] Western Blot experiments were also performed on 9 cell and gene combinations.

[0255] (1) The results of screening the expression of target proteins under different cell gene combinations are shown in Figure 5-Figure 6 .

[0256] Figure 5 Describes the results of GST activity detection experiments using three cell types and vectors containing three different tags. Each combination has four data points. The sample sources of these data points are Figure 2-Figure 4 The samples used in the SDS-PAGE experiment were from the same test tube. The "*" on the line connecting the two groups of data in the figure indicates that the data are significantly different, where * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.005.

[0257] Conclusion: GST activity results showed that the best combinations were BL21 and GST, STK and GST, and TE and NuSA-GST. WB results showed that the best combinations were BL21 and NuSA-GST, BL21 and GST, and TE and DsbA-GST. The inconsistency of the two experimental results may be caused by different protein sample treatment methods. Comprehensively considering that the BL21GST combination had the best results in both experiments, BL21GST was the ideal screening result among the 9 combinations.

[0258] Figure 6 Western Blot results of three types of cells and vectors containing three different tags. The source of samples for Western Blot experiments and Figure 2-Figure 4 The samples used in the SDS-PAGE experiment are consistent, and the abbreviation information in the sample name is consistent with the number information description. From this figure, it can be concluded that the three combinations of BL21NuSA-GST, BL21GST, and TE DsbA-GST are the best.

[0259] according to Figure 2-Figure 6 The experimental conclusion obtained was that among the 9 combinations, the combination of BL21 and a vector containing a GST tag was the best.

[0260] (2) The effects of different inducer (IPTG) concentrations on the expression of target protein in BL21-pET-28a-GST-BONTA cell gene combinations are shown in Figure 7-Figure 9 .

[0261] Figure 7 The results of the GST activity detection experiment of the target protein of the BL21GST combination at different inducer concentrations are described. Except for the blank (0mM inducer), there are 3 data for each concentration. The sample for activity detection is bacterial lysate, and the lysis method is to add lysozyme at a final concentration of 1mg / mL, freeze and thaw repeatedly 4 times, and take the supernatant as the bacterial lysate. From this figure, it can be concluded that there is a significant difference between different inducer concentrations in the BL21GST combination and 0mM, and there is no significant difference between different inducer concentrations except 0mM. The content of the target protein induced by the inducer at a concentration of 0.4-1.0mM is better, among which the concentration of 0.4mM is the best.

[0262] Figure 8 Describes the Western Blot results of the target protein under different inducer concentrations of the BL21GST combination. The numbers in the second row of the sample name indicate the inducer concentration in mM. ① indicates Figure 7Sample No. 1 of the three parallel samples tested for GST activity. From this figure, it can be concluded that the protein expression induced by different inducer concentrations in the BL21GST combination is significantly different from that induced by 0mM, and the protein expression level as a whole tends to decrease slightly with increasing inducer concentration.

[0263] Fig. 9 for Figure 8 Frame part of the target band and use gelpro software to analyze the grayscale value. The high and low grayscale values ​​reflect the high and low protein expression levels. The conclusions drawn from this figure are similar to Figure 8 Consistent.

[0264] In addition to the cell types involved in the embodiments, the present invention has also been verified by vector transfection for other cell types mentioned in the present invention, but the effects are not ideal and are not suitable for achieving the purpose of the present invention.

[0265] Example 2 Purification process of BL21-pET-28a-MGSS-6His-GST-thrombin-bonta (vector number 2)

[0266] The specific conditions of some steps in this implementation are as follows:

[0267] 1. Centrifugation, homogenization, clarification

[0268] The fermented broth was centrifuged at 14000 r / min to obtain bacterial sludge. The bacterial sludge was mixed with Ni-NTA buffer (20 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.5) were mixed and dispersed in a ratio of 1:10, and crushed using a homogenizer at 800 bar. The supernatant was clarified by a nine-year-old 0.45 um membrane package with a transmembrane pressure of 0.3 bar to obtain a supernatant.

[0269] 2. Ni-NTA affinity chromatography capture

[0270] The supernatant was loaded onto a Ni-NTA column (Bocron) at a flow rate of 100 cm / h and a buffer (20 mM Na 2 HPO 4 , 150 mM NaCl, pH 7.5), eluent (200 mM imidazole, 20 mM Na 2 HPO 4 , 150 mM NaCl, pH 8.0), gradient elution, 30% elution, 100% elution to the desired target protein.

[0271] 3. Hydrophobic chromatography

[0272] The sample obtained by eluting 100% through the Ni-NTA column was added with 0.7 mol ammonium sulfate and loaded onto a hydrophobic column (Bailink) at a flow rate of 100 cm / h and a buffer (20 mM Na 2 HPO 4 , 0.7 mol(NH 4 ) 2 SO 4 , pH 9.0), eluent (20 mM Na 2 HPO 4 , pH 9.0), gradient elution, 30%, 50%, 100%. 50% obtained the desired target protein sample.

[0273] 4. G25 fluid change

[0274] The hydrophobic sample was loaded onto a G25 column (GE) at a volume less than 30% of the column volume, with a flow rate of 100 cm / h and a mobile phase of 20 mM Tris-HCl, pH 8.0, and the elution peak was collected.

[0275] 5. SP ion exchange chromatography

[0276] The sample after G25 liquid exchange was loaded onto the SP column at a flow rate of 200 cm / h, equilibration solution (20 mM Tris-HCl, pH 8.0), eluent (20 mM Tris-HCl, 1 mol NaCl, pH 8.0), 10% washing, 15% elution to obtain the sample, and 2 mol NaCl for regeneration.

[0277] 6. Thrombin cleavage

[0278] Add bovine thrombin (Shanghai Yuanye) to the sample at a ratio of 6U-200U:1mg to the target protein, and perform overnight digestion in a 37°C constant temperature incubator. Run the gel to observe whether the digestion is complete. If not, extend the digestion time.

[0279] 7. Removal of GST tag by affinity chromatography

[0280] The digested sample was loaded onto a GST column (Yisheng) at a flow rate of 30 cm / h, with equilibration solution (PBS, pH 7.2) and elution solution (20 mM Tris-HCl, 50 mM reduced glutathione, pH 8.0). The GST tag was bound to the column, and the target protein with the tag cut off could not bind to the column. The flow-through peak was collected.

[0281] 8.G200 molecular sieve

[0282] The sample was ultrafiltered and concentrated using a 30kD ultrafiltration membrane (Jiuling) with a cross-mold pressure of 0.5 bar to a protein concentration of 1-2 mg / mL, and then filtered through a PG200 molecular sieve (Bailinke) at a flow rate of 20 cm / h. The buffer was 20 mM sodium lactate, pH 7.0. Different elutions were collected and run on a gel to determine the peak position of the target protein.

[0283] The purpose of this example is to optimize the purification process and obtain a high-purity BoNT / A sample, focusing on the composition and sequence of purification unit operations. Each experimental process and each unit operation are performed under almost the same experimental conditions, including but not limited to the composition of the loading buffer, elution conditions, pH value, filler loading, loading flow rate, column height, column diameter, column volume, etc. The specific results are as follows:

[0284]

[0285]

[0286] Taking the result of process 7 as an example, Fig.10 The results of SDS-PAGE experiments on samples containing the target protein after removal of the GST tag and PG200 molecular sieve chromatography are described. From this figure, it can be concluded that a single band can be obtained after PG200 molecular sieve chromatography of the target protein.

[0287] Molecular design rationality verification:

[0288] Clostridial neurotoxins are synthesized as single-chain polypeptides that are post-translationally modified by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often referred to as the activation site, located between the cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the most active form of the neurotoxin.

[0289] In order to achieve the removal of the GST tag (related to step 6 of the purification process), the thrombin recognition site LVPRG / S (related to thrombin) was added after the GST protein; in order to achieve the activation of the single-chain botulinum toxin, the thrombin recognition site LVPRG / S was knocked into the loop region (loop) connecting the light chain and heavy chain of the botulinum toxin, and bovine thrombin and porcine thrombin were used to verify the cutting effect. Specific conditions:

[0290]

[0291] The cleavage experiment found that thrombin could very efficiently cleave the LC and HC connection region, but could not effectively remove the N-terminal GST tag.

[0292] It is speculated that the GST-tagged protein and the botulinum toxin molecule are too closely connected in space, resulting in the inability to effectively cut GST-LC even with a high activity equivalent of 200U of thrombin.

[0293] Therefore, a flexible linker GGGSGGGS was added between GST and the botulinum toxin molecule. pET-28a-MGSS-6His-GST-GGGSGGGS-thrombin-GGGS-bonta (vector sequence number 7) was constructed and transduced into BL21 (DE3) competent cells. After expression and purification, it was found that thrombin cleavage not only effectively cleaved the connection region between the light chain LC and the heavy chain HC of the botulinum toxin, but also effectively cleaved the N-terminal GST tag protein. The amount of thrombin used was reduced by 10 times. The results are as follows: Fig.11 .

[0294] Fig.11 The results of the SDS-PAGE experiment after the target protein was digested with thrombin are described. In this experiment, lane 2 is a sample without thrombin (i.e., it only contains the target protein), lanes 3-8 are samples digested with porcine thrombin, and lanes 9-14 are samples digested with bovine thrombin. Among the above lanes, except for lanes 8 and 14 which are non-reduced SDS-PAGE, the remaining lanes are reduced SDS-PAGE. The letter Z in the sample name represents porcine thrombin, the letter N represents bovine thrombin, and the number after the letter represents the activity of the enzyme: 1 is 2U, 2 is 5U, 3 is 10U, 4 is 20U, and 5 is 30U. The digestion condition is overnight digestion at 30°C. The results in the figure show that after thrombin digestion, bands that meet the theoretical molecular weight appear, which are ① only the N-terminal digestion site is cut, leaving a 150Kda band of light chain and heavy chain connected by disulfide bonds and a GST tag band of about 30Kda; ② only the digestion site between the light chain and heavy chain of the target protein is cut, leaving a 100Kda heavy chain band and a light chain band containing a GST tag of about 80Kda; ③ Both digestion sites are cut, leaving a 100Kda heavy chain band, a 50Kda light chain band and a 30Kda tag band. Similarly, it can be observed in the results that with the increase in the amount of thrombin used, the content of the 30Kda GST tag band, the thrombin band, the 50Kda light chain band, the 80Kda light chain band containing the GST tag and the 100Kda heavy chain band also gradually increases, and the content of the 150Kda band that only cuts the N-terminal tag gradually decreases.

[0295] Fig.12The results of SDS-PAGE experiments after thrombin digestion of target proteins from different sample sources (different samples after purification) are described. In this experiment, the samples used in lanes 2-6 were obtained from NTA purification columns. The target protein contained a GST tag and had a theoretical molecular weight of about 180Kda. Lane 4 was a sample without enzyme addition, and lanes 2 and 3 were the results of non-reduced SDS-PAGE experiments after adding 25U and 50U bovine thrombin to digest the samples, respectively. Lanes 5 and 6 were the results of reduced SDS-PAGE experiments of the corresponding samples in lanes 2 and 3; the samples used in lanes 7-13 were obtained from SP columns and then hydrophobic columns. The target protein did not contain a GST tag and had a theoretical molecular weight of 150Kda. Lane 10 was a sample without enzyme addition, and lanes 7-10 were the results of non-reduced SDS-PAGE experiments after adding different bovine thrombin digestion activities. Lanes 11-13 were samples of reduced SDS-PAGE experiments of the corresponding samples in lanes 7-10. Lanes 14 and 15 are the results of reduced SDS-PAGE of pure porcine thrombin and pure bovine thrombin samples. The results in the figure show that after the target protein containing the GST tag is digested, a band that conforms to the theoretical molecular weight is obtained, and all the digestion sites are cut, and the following bands are obtained: ①150Kda bands of light chain and heavy chain connected by disulfide bonds, ②100Kda heavy chain, ③50Kda light chain, ④ newly introduced thrombin band, ⑤ cut-off GST tag band of about 30Kda. After the target protein without the GST tag is digested, bands that conform to the theoretical expected molecular weight are obtained, namely ①150Kda uncut target band and bands of light chain and heavy chain connected by disulfide bonds, ②100Kda heavy chain, ③50Kda light chain.

[0296] After GST affinity chromatography to remove the tag and G200 molecular sieve, high-purity recombinant BONTA ( Fig.13 ).

[0297] Fig.13 The results of the SDS-PAGE experiment after the target protein without GST tag was digested with thrombin are described. Lanes 1 and 2 are the results of the non-reduced SDS-PAGE experiment before and after the target protease cleavage, respectively, and lanes 3 and 4 are the results of the reduced SDS-PAGE experiment of the corresponding samples in lanes 1 and 2. The results in the figure show that after the target protein was digested, bands with the expected molecular weight were obtained, which are ①150Kda bands of light chain and heavy chain connected by disulfide bonds, ②100Kda heavy chain, and ③50Kda light chain.

[0298] The above results show that the molecular structure is consistent with the theoretical design.

[0299] It should be noted that the above results are only used to illustrate the present invention, not to limit the present invention. Further improvements or replacement of conventional technical means by those skilled in the art based on the technical solution disclosed in the present invention are all within the scope of protection of the present invention.

Claims

1. A recombinant genetically engineered bacterium expressing botulinum toxin type A, characterized in that: It comprises a pET-28a vector, wherein the pET-28a vector comprises a gene fragment 1 expressing a GST tag, a gene fragment 2 expressing a thrombin recognition site, and a gene fragment 3 expressing BoNT / A; the amino acid sequence of the GST is SEQ ID NO.1; the amino acid sequence of the thrombin recognition site is SEQ ID NO.2; and the amino acid sequence of the BoNT / A is SEQ ID NO.3; The pET-28a vector also includes gene fragment 5 and gene fragment 6 expressing a connecting peptide; the connecting peptide is located at both ends of the thrombin recognition site LVPRGS expressed by the gene fragment 2, and the connecting peptide is (GGGS)n, wherein n is 1, 2 or 3; The pET-28a vector also includes a gene fragment 7 expressing MGSS and a gene fragment 8 expressing a histidine tag; the histidine tag is 6His; The order of the gene fragments on the pET-28a vector is gene fragment 7-gene fragment 8-gene fragment 1-gene fragment 5-gene fragment 2-gene fragment 6-gene fragment 3; The BoNT / A light chain and heavy chain connecting region includes another thrombin recognition site with an amino acid sequence of SEQ ID NO. 2, and the expression gene of the thrombin recognition site on the BoNT / A is gene segment 4, and the gene segment 4 is located in the connecting region of the gene segment 3 encoding the light chain segment and the heavy chain segment; The recombinant genetic engineering bacteria is Escherichia coli BL21.

2. The recombinant genetically engineered bacterium according to claim 1, characterized in that: The n value of the connecting peptide expressed by gene fragment 5 is 2, and it is located at the N-terminus of the thrombin recognition site LVPRGS expressed by gene fragment 2; the n value of the connecting peptide expressed by gene fragment 6 is 1, and it is located at the C-terminus of the thrombin recognition site LVPRGS expressed by gene fragment 2.

3. The recombinant genetically engineered bacterium according to claim 2, characterized in that: The nucleotide sequence of the gene fragment 5 is the nucleotide sequence shown in SEQ ID NO.8 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.8 and expresses the same amino acid; the nucleotide sequence of the gene fragment 6 is the nucleotide sequence shown in SEQ ID NO.9 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.9 and expresses the same amino acid.

4. The recombinant genetically engineered bacterium according to claim 1, characterized in that: The nucleotide sequence of the gene fragment 7 is SEQ ID NO.10 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.10 and expresses the same amino acid; the nucleotide sequence of the gene fragment 8 is SEQ ID NO.11 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.11 and expresses the same amino acid.

5. The recombinant genetically engineered bacterium according to any one of claims 1 to 4, characterized in that: The nucleotide sequence of the gene fragment 1 expressing the GST tag is the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.4 and expresses the same amino acids.

6. The recombinant genetically engineered bacterium according to claim 5, characterized in that: The gene fragment 2 or gene fragment 4 expressing the thrombin recognition site is selected from the nucleotide sequence shown in any one of SEQ ID NO.5-6 or a nucleotide sequence that has more than 80% identity with the nucleotide sequence shown in any one of SEQ ID NO.5-6 and expresses the same amino acid.

7. The recombinant genetically engineered bacterium according to claim 6, characterized in that: The nucleotide sequence of the gene fragment 3 expressing BoNT / A is the nucleotide sequence shown in SEQ ID NO.7 or a nucleotide sequence that has more than 80% identity with SEQ ID NO.7 and expresses the same amino acid.

8. The recombinant genetically engineered bacterium according to claim 1, characterized in that: The pET-28a vector includes the nucleotide sequence shown in SEQ ID NO.10-SEQ ID NO.11-SEQ ID NO.4-SEQ ID NO.8-SEQ ID NO.5-SEQ ID NO.9-SEQ ID NO.7 or a nucleotide sequence with more than 80% identity and expressing the same amino acid.

9. The recombinant genetically engineered bacterium according to claim 1, characterized in that: The insertion site of the pET-28a vector is selected from any one or two of EcoRI, NcoI, BstEⅡ, SphⅠ, NruⅠ, PasⅠ, PmeⅠ, BamHI or RsrⅡ.

10. The recombinant genetically engineered bacterium according to claim 9, characterized in that: The insertion site of the pET-28a vector is EcoRI / NcoI.

11. The method for constructing a recombinant genetically engineered bacterium according to any one of claims 1 to 10, characterized in that: The method comprises inserting each gene fragment into the pET-28a vector in sequence.

12. Use of the recombinant genetically engineered bacteria according to any one of claims 1 to 10 in the preparation of botulinum toxin type A.

13. A method for preparing botulinum toxin type A, characterized in that: The method comprises using the recombinant genetic engineering bacteria described in any one of claims 1 to 10 for expression.

14. The preparation method according to claim 13, characterized in that: The expression conditions are as follows: the recombinant genetically engineered bacteria are inoculated into a culture medium and cultured at 35-40° C. and induced at 16-25° C.; the induction is achieved by adding an inducer, and the concentration of the inducer added is 0-1 mM.

15. The preparation method according to claim 14, characterized in that: The inducing agent is added at a concentration of 0.4-1 mM.

16. The preparation method according to claim 15, characterized in that: The inducer was added at a concentration of 0.4 mM.

17. The preparation method according to claim 14, characterized in that: The inducing agent is IPTG, lactose or L-arabinose.

18. The preparation method according to claim 14, characterized in that: It also includes the purification of botulinum toxin type A, and the purification includes any one or more of centrifugation, homogenization, clarification, NTA affinity chromatography, hydrophobic chromatography, G25 fluid exchange, SP ion exchange, thrombin enzyme cleavage, GST affinity chromatography, and G200 molecular sieve.

19. The preparation method according to claim 18, characterized in that: The purification further comprises: NTA affinity chromatography, hydrophobic chromatography, G25 liquid exchange and SP ion exchange.

20. The preparation method according to claim 19, characterized in that: The purification also includes: any one or more of thrombin digestion, GST affinity chromatography, and G200 molecular sieve.

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