Preparation method and application of large intestine expression recombinant A-type botulinum toxin

Abstract

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

Description

Technical Field

[0001] The present invention relates to the fields of bioengineering and pharmaceutical engineering, and specifically relates to an expression vector for expressing recombinant botulinum neurotoxin type A, an engineered bacterium, a method for preparing recombinant botulinum neurotoxin type A, and its applications, etc. Background Art

[0002] Natural botulinum neurotoxin (BoNT) is one of the most toxic substances in the world produced by anaerobic Clostridium botulinum. Seven serotypes of BoNT have been discovered so far, namely types A, B, C, D, E, F, and G. Among them, botulinum neurotoxin of type A (BoNT / A) is the most commonly used in medical aesthetics and clinical treatment.

[0003] Currently, BoNT / A is mainly derived from natural extraction. Although its process is mature and has a long application history, it has limitations such as complex preparation processes, containing a large amount of non-BoNT / A components, and potential biosafety risks in the production process. In contrast, recombinant botulinum neurotoxin type A obtained by recombinant technology has the following obvious advantages: 1) Safety: Recombinant BoNT / A is obtained by fermenting and culturing recombinant engineered bacteria. It has low toxicity before activation, eliminating the potential biosafety risks associated with extracting BoNT / A from Clostridium botulinum. 2) Low immunogenicity: Recombinant BoNT / A has higher purity, reducing the risk of generating drug-resistant antibodies. 3) Stable efficacy: Recombinant BoNT / A directly expresses a single botulinum toxin protein through genetic recombination technology. The highly efficient and controllable preparation process ensures high consistency of products between batches.

[0004] However, the preparation of recombinant BoNT / A requires a higher technical level. BoNT / A is a dimer protein of approximately 150 kDa composed of a light chain (LC, 50 kDa) and a heavy chain (HC, 100 kDa) linked by a disulfide bond. Due to its large molecular weight and being a toxic protein, it is difficult to achieve soluble expression directly in Escherichia coli under normal circumstances. In addition, the BoNT / A expressed directly in E. coli is a single-chain protein and needs to be further digested by enzymes to form an LC-HC dimer protein to activate its toxicity. The following two preparation processes of recombinant BoNT / A have been reported:

[0005] One is to express LC and HC separately in Escherichia coli in the form of inclusion bodies, and then obtain recombinant BoNT / A through renaturation and assembly. The recombinant BoNT / A obtained by this method has the same sequence as natural BoNT / A, but the inclusion body protein needs to undergo complex denaturation and renaturation processes to restore its biological activity, and the renaturation rate is usually low; in addition, the renaturation and assembly processes are complex, making it difficult to ensure batch-to-batch differences, which is not conducive to commercial-scale production and application.

[0006] Second, the soluble expression of recombinant BONT / A protein in E. coli was achieved by introducing a tag protein and a cleavable Linker, and then recombinant BONT / A was obtained through purification and cleavage. The soluble expressed recombinant BONT / A can maintain its native conformation and biological activity, and the activation of toxicity after cleavage can greatly improve the safety of production and storage, which is beneficial to commercial production and application. However, the recombinant BONT / A obtained by this method usually has multiple foreign amino acid residues, one is the residue of the cleavable Linker at the N-terminus of LC, the second is the residue of the cleavable Linker between LC and HC, and the third is the residue of the cleavable Linker at the C-terminus of HC. All the reported soluble expressed recombinant BONT / A contain at least two or more foreign amino acid residues.

[0007] The prior art has disclosed different methods for preparing recombinant BONT / A E. coli. For example, Patent Document 1 (CN116440281A, publication date: July 18, 2023) discloses a botulinum toxin protein composition and its preparation method. The recombinant botulinum toxin protein is composed of at least one dimer structure formed by polypeptides after protease cleavage, and it contains a structural region with a first protease cleavage site and a structural region with a second protease cleavage site. Patent Document 2 (CN118006523A, publication date: May 10, 2024) discloses a recombinant genetic engineering bacterium expressing botulinum toxin type A, including 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; another thrombin recognition site with the amino acid sequence of SEQ ID NO.2 is included in the connection region between the light chain and the heavy chain of BoNT / A. It can be seen that the methods for preparing recombinant BONT / A E. coli in the prior art all contain a cleavage region for enzymatic cleavage recognition between the heavy chain and the light chain, and the obtained recombinant BONT / A contains multiple foreign amino acid residues.

[0008] Therefore, there is an urgent need in the art to develop an E. coli expression vector, engineering bacterium, and preparation method of recombinant BONT / A with as few introduced or no introduced foreign amino acids as possible through novel molecular design and preparation processes, and it has significant research significance and application value to obtain recombinant BONT / A with high purity, strong toxicity, low residue, high efficiency and controllability, and easy to achieve commercial-scale production. Summary of the Invention

[0009] In view of the deficiencies of the prior art, the present invention efficiently obtains a recombinant botulinum neurotoxin serotype A (reBONT / A) with stronger toxicity and lower immunogenicity through novel molecular design and preparation processes. Specifically, the present invention provides a nucleic acid expression cassette for recombinant botulinum neurotoxin serotype A (BONT / A), which comprises a tag, a cleavage site, and a nucleic acid sequence of botulinum neurotoxin serotype A (BONT / A), and no exogenous protease recognition site is introduced between the light chain and the heavy chain of BONT / A. Further, the present invention provides a recombinant vector, a recombinant bacterium, a protein encoded and expressed, a method for preparing BONT / A using the same, and its applications, etc.

[0010] In the first aspect of the present application, there is provided a nucleic acid expression cassette for recombinant botulinum neurotoxin serotype A (BONT / A), which is characterized in that it sequentially comprises the following elements from the 5'-end to the 3'-end: nucleic acid encoding a tag protein, nucleic acid encoding a cleavage site, and a BONT / A nucleic acid sequence, wherein no exogenous protease recognition site is introduced between the light chain and the heavy chain of the BONT / A.

[0011] Further, it is characterized in that the tag protein comprises one or more of HIS tag, GSTs tag, MBP tag, NusA tag, SUMO tag, and TrxA tag; and the cleavage site comprises one or more of HRV 3C cleavage site and enterokinase cleavage site.

[0012] Further, it is characterized in that the nucleic acid expression cassette comprises the following (1) or (2):

[0013] (1) reBONT / A gene: sequentially comprising the following nucleic acid elements from the 5'-end to the 3'-end: HIS tag - GSTs tag - HRV3C cleavage site - BONT / A; or

[0014] (2) reBONT / A-1 gene: sequentially comprising the following nucleic acid elements from the 5'-end to the 3'-end: HIS tag - GSTs tag - Linker - enterokinase cleavage site - BONT / A.

[0015] Further, it is characterized in that the amino acid sequence of the HIS tag can be MGSS(H) n, where n is an integer from 6 to 10. Preferably, the nucleotide sequence of the HIS tag is as shown in SEQ ID NO.1, and the amino acid sequence of the HIS tag is as shown in SEQ ID NO.2; the nucleotide sequence of the GSTs tag is as shown in SEQ ID NO.3, and the amino acid sequence of the GSTs tag is as shown in SEQ ID NO.4; the nucleotide sequence of the HRV 3C cleavage site is as shown in SEQ ID NO.5, and the amino acid sequence of the HRV 3C cleavage site is as shown in SEQ ID NO.6; the amino acid sequence of the Linker can be (GGGGSGGGS) n , where n is an integer ≥ 1. Preferably, n = 1. The nucleotide sequence of the Linker is as shown in SEQ ID NO.7, and the amino acid sequence of the Linker is as shown in SEQ ID NO.8; the nucleotide sequence of the enterokinase cleavage site is as shown in SEQ ID NO.9, and the amino acid sequence of the enterokinase cleavage site is as shown in SEQ ID NO.10; the nucleotide sequence of the BONT / A is as shown in SEQ ID NO.11, and the amino acid sequence of the BONT / A is as shown in SEQ ID NO.12.

[0016] Further, it is characterized in that the nucleotide sequence of the reBONT / A is as shown in SEQ ID NO.13, and the amino acid sequence of the reBONT / A is as shown in SEQ ID NO.14; the nucleotide sequence of the reBONT / A-1 is as shown in SEQ IDNO.15, and the amino acid sequence of the reBONT / A-1 is as shown in SEQ ID NO.16.

[0017] The second aspect of the present application provides a recombinant vector, which is characterized by comprising the recombinant botulinum neurotoxin type A (BONT / A) nucleic acid expression cassette.

[0018] Further, it is characterized in that the recombinant vector is a pET series vector; preferably, the pET series vector is pET-28a.

[0019] The third aspect of the present application provides a recombinant expression bacterium, which is characterized by comprising the recombinant botulinum neurotoxin type A (BONT / A) nucleic acid expression cassette, or the recombinant vector.

[0020] Furthermore, it is characterized in that the recombinant expression bacterium is Escherichia coli; preferably, the Escherichia coli includes E. coli BL21(DE3), E. coli Origami B(DE3), E. coli Rosetta Blue(DE3), E. coli C41(DE3); more preferably, the Escherichia coli is the E. coli C41(DE3) strain.

[0021] The fourth aspect of the present application provides a recombinant botulinum neurotoxin type A (BONT / A) protein, which is characterized in that it is obtained by encoding and expressing with the recombinant botulinum neurotoxin type A (BONT / A) nucleic acid expression cassette.

[0022] The fifth aspect of the present application provides a method for constructing the recombinant expression bacterium, which is characterized in that the recombinant botulinum neurotoxin type A (BONT / A) nucleic acid expression cassette is used to construct a recombinant expression plasmid by restriction enzyme digestion and ligation or one-step cloning, and then the recombinant expression plasmid is transformed into Escherichia coli competent cells by heat shock method.

[0023] The sixth aspect of the present application provides a method for preparing recombinant botulinum neurotoxin type A (BONT / A) expressed in Escherichia coli, which is characterized in that it includes the steps of fermenting with the recombinant expression bacterium, digesting with enterokinase to cut open the single-chain BONT / A light chain and heavy chain connection region, and the cut light chain and heavy chain form a double-chain BONT / A protein, and purifying to obtain the recombinant BONT / A; preferably, preliminary purification by NTA affinity chromatography is also included before enterokinase digestion.

[0024] Furthermore, it is characterized in that the method includes obtaining a crude purified solution by homogenizing, centrifuging and filtering the cells of the recombinant expression bacterium; the crude purified solution is subjected to fine purification, including any one or more of NTA affinity chromatography, GST affinity chromatography, protease digestion and DEAE ion exchange chromatography.

[0025] Furthermore, it is characterized in that the fine purification includes any one of the following: (1) NTA affinity chromatography - protease digestion - NTA affinity chromatography - DEAE ion exchange chromatography; or (2) NTA affinity chromatography - GST affinity chromatography - protease digestion - GST affinity chromatography - DEAE ion exchange chromatography.

[0026] Furthermore, it is characterized in that the protease digestion includes any one or more of HRV 3C protease digestion or enterokinase digestion.

[0027] Furthermore, it is characterized in that for the preparation of the recombinant protein expressed by the reBONT / A gene, HRV 3C enzyme is used to cleave the HRV 3C enzyme recognition site to separate the HIS-GSTs tag and the single-chain BONT / A protein, and enterokinase is used to cleave the BONT / A light chain and heavy chain connection region to form a double-chain BONT / A protein; the order of the HRV 3C enzyme cleavage and enterokinase enzyme cleavage can be any of the following: 1) The HRV 3C enzyme cleavage and enterokinase enzyme cleavage are carried out one by one; 2) The HRV 3C enzyme cleavage and enterokinase enzyme cleavage are carried out simultaneously; preferably, the HRV 3C enzyme cleavage and enterokinase enzyme cleavage are carried out simultaneously; the double-chain BONT / A obtained after further purification of the enzyme cleavage product has only two foreign amino acids remaining at the N-terminus of the BONT / A light chain.

[0028] Furthermore, it is characterized in that for the preparation of the recombinant protein expressed by the reBONT / A-1 gene, only enterokinase digestion is required to separate the HIS-GSTs tag and cleave the BONT / A light chain and heavy chain connection region to form a double-chain BONT / A protein; the double-chain BONT / A protein sequence obtained after further purification of the enzyme cleavage product is exactly the same as that of natural BONT / A, without any foreign amino acid residues.

[0029] The seventh aspect of the present application provides the use of the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette, the recombinant vector, the recombinant expression bacterium, the recombinant botulinum toxin type A (BONT / A) protein, the construction method of the recombinant expression bacterium, and the preparation method of the recombinant botulinum toxin type A (BONT / A) expressed in the large intestine in the preparation of BONT / A.

[0030] The eighth aspect of the present application provides the use of the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette, the recombinant vector, the recombinant expression bacterium, the recombinant botulinum toxin type A (BONT / A) protein, the construction method of the recombinant expression bacterium, and the preparation method of the recombinant botulinum toxin type A (BONT / A) expressed in the large intestine in the medical and cosmetic fields; preferably, the use is a medical aesthetic product or a clinical drug.

[0031] The nucleic acid expression cassette, recombinant vector, and preparation method of BONT / A of the present invention have the following excellent technical effects:

[0032] 1. Natural BONT / A is a double-stranded structure composed of a light chain and a heavy chain. Generally, obtaining recombinant BONT / A requires introducing an exogenous protease recognition site between the light chain and the heavy chain of single-chain BONT / A and then cleaving the light chain and the heavy chain through enzymatic digestion. The present invention for the first time discovers and determines that there is no need to additionally introduce an exogenous protease recognition site between the light chain and the heavy chain of BONT / A. Only enterokinase can effectively cleave the connection between the light chain and the heavy chain in natural single-chain BONT / A to form double-stranded BONT / A, and its toxicity is significantly higher than that of recombinant BONT / A obtained by introducing an exogenous enzymatic cleavage site at the connection between the light chain and the heavy chain. Under the same conditions, its toxicity is 5 times higher than that of traditional recombinant botulinum toxin type A with double protease recognition sites.

[0033] 2. Conventional E. coli expression of soluble recombinant botulinum toxin type A requires introducing multiple exogenous amino acids at more than two positions. For the recombinant protein expressed by the reBONT / A gene of the present invention, only two exogenous amino acids remain at the N-terminus of the light chain of BONT / A in the double-stranded BONT / A obtained after further purification of the enzymatic digestion product. For the recombinant protein expressed by the reBONT / A-1 gene of the present invention, the protein sequence of the double-stranded BONT / A obtained after further purification of the enzymatic digestion product is exactly the same as that of natural BONT / A, without any residual exogenous amino acids. It can be seen that the recombinant botulinum toxin type A prepared by the present invention does not introduce exogenous amino acids or only introduces two amino acids at one position, ensuring the maximum consistency with natural botulinum toxin type A to the greatest extent, and the obtained BONT / A has low immunogenicity.

[0034] 3. The present invention also optimizes the preparation process of recombinant BONT / A, and obtains high-purity and low-residual recombinant BONT / A only through 3 steps of chromatography and 1 step of enzymatic digestion. It can efficiently obtain recombinant botulinum toxin type A with a purity of more than 97%, and LD 50 is 10 pg / animal, and under the same conditions, its toxicity is 5 times higher than that of traditional recombinant botulinum toxin type A with double protease recognition sites. Description of the Drawings

[0035] Figure 1 This is the amino acid sequence alignment of BONT / A with different molecular designs of the present invention, where BONT_A is the amino acid sequence of natural BONT / A, reBONT_A (i.e., reBONT / A) and reBONT_A_1 (i.e., reBONT / A-1) are the amino acid sequences of reBONT / A of the present invention, and CN11644028 (i.e., CN116440281A) and CN11800652 (i.e., CN118006523A) are the reported amino acid sequences of recombinant BONT / A.

[0036] Figure 2 This is the Western blot result of the expression of reBONT / A fusion protein under different induction conditions of the present invention, whereFigure 2 In a, it shows the expression of reBONT / A fusion protein under different induction temperatures and inducer concentrations. Figure 2 In b, it shows the expression of reBONT / A fusion protein under different induction durations.

[0037] Figure 3 This is the SDS-PAGE result of the preliminary purification of the reBONT / A fusion protein of the present invention. Among them, lane 1 is the protein Marker, and lane 2 is the reBONT / A fusion protein.

[0038] Figure 4 This is the SDS-PAGE result of the cleavage of the reBONT / A fusion protein of the present invention. Among them, lane 1 is the protein Marker, lane 2 is the reBONT / A fusion protein, lane 3 is the BONT / A single-chain protein and HIS-GSTs tag protein after cleavage by 3C enzyme, lane 4 is the BONT / A double-chain protein and HIS-GSTs tag protein after cleavage by enterokinase, and lane 5 is the result of the dissociation of the double chain of the sample in lane 4 under reducing conditions.

[0039] Figure 5 This is the SDS-PAGE result of the purification of the reBONT / A fusion protein of the present invention. Among them, lane 1 is the protein Marker, lane 2 is the purified BONT / A double-chain protein, and lane 3 is the HIS-GSTs tag protein.

[0040] Figure 6 This is the SDS-PAGE result of the purification of the reBONT / A double-chain protein of the present invention. Among them, lane 1 is the protein Marker, lane 2 is the reBONT / A double-chain protein with a purity greater than 97% after purification, and lane 3 is the verification result of the dissociation of the double chain under reducing conditions. Detailed implementation methods

[0041] The present invention will be further described in detail below with reference to the accompanying drawings and implementation cases. However, the protection scope of the present invention is not limited to the content described. In the implementation cases, conventional molecular biology and protein engineering methods are mainly used, and these methods are well-known to those of ordinary biotechnologists in the field. The specific reagents, plasmids, strains, culture media, instruments, etc. used in the examples are all commercially available in the field.

[0042] Example 1 Design and construction of the single-chain recombinant BONT / A of the modified botulinum toxin type A of the present invention

[0043] The recombinant BONT / A of the present invention can be in two forms, as follows

[0044] (1) reBONT / A: HIS tag - GSTs tag - HRV 3C protease cleavage site - BONT / A.

[0045] (2) reBONT / A-1: HIS tag - GSTs tag - Linker - Enterokinase cleavage site - BONT / A.

[0046] (1) Design and construction of reBONT / A

[0047] 1. Design of reBONT / A. Starting from the 5'-end, reBONT / A successively contains:

[0048] a. The nucleotide sequence encoding tag protein 1 (histidine tag, i.e., HIS tag) is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2;

[0049] b. The nucleotide sequence encoding tag protein 2 (glutathione S-transferase, i.e., GSTs tag) is shown in SEQ ID NO.3, and the encoded amino acid sequence is shown in SEQ ID NO.4;

[0050] c. The nucleotide sequence encoding the protease cleavage site (i.e., HRV 3C cleavage site) is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6;

[0051] d. The nucleotide sequence encoding the complete BONT / A is shown in SEQ ID NO.11, and the encoded amino acid sequence is shown in SEQ ID NO.12.

[0052] The nucleotide sequence of reBONT / A constructed in the present invention is shown in SEQ ID NO.13, and the amino acid sequence of reBONT / A is shown in SEQ ID NO.14.

[0053] 2. Construction of a recombinant expression plasmid containing the sequences of step 1

[0054] Entrust a gene synthesis company to synthesize the reBONT / A gene, introduce Nco I and Xho I cleavage sites at both ends, and then insert it into the Nco I and Xho I sites in the pET28a plasmid vector by enzymatic digestion and ligation.

[0055] (2) Design and construction of reBONT / A-1

[0056] Referring to the construction process of reBONT / A described above (1), except for the Linker and the kinase cleavage site, the sequences of other elements are the same. Among them, the nucleotide sequence of the Linker is shown in SEQ ID NO.7, the amino acid sequence of the Linker is shown in SEQ ID NO.8, the nucleotide sequence of the enterokinase cleavage site is shown in SEQ ID NO.9, and the amino acid sequence of the enterokinase cleavage site is shown in SEQ ID NO.10. The nucleotide sequence of reBONT / A-1 constructed in the present invention is shown in SEQ ID NO.15, and the amino acid sequence of reBONT / A-1 is shown in SEQ ID NO.16. The recombinant plasmid pET28a-reBONT / A-1 containing reBONT / A-1 was constructed.

[0057] Align the amino acid sequences of BONT / A designed with different molecules, such as Figure 1 , where BONT_A is the natural BONT / A amino acid sequence, reBONT_A and reBONT_A_1 are the recombinant BONT / A amino acid sequences of the present invention, CN116440281A (i.e., Figure 1 CN11644028 in Figure 1 ) and CN118006523A (i.e., Figure 1 CN11800652 in Figure 1 are the reported recombinant BONT / A amino acid sequences. It can be seen that for the recombinant protein expressed by the reBONT / A (i.e., reBONT_A in Figure 1 ) gene, the HRV 3C enzyme is used to cleave the HRV 3C recognition site to separate the HIS-GSTs tag and the single-chain BONT / A protein, and the enterokinase is used to cleave the BONT / A light chain and heavy chain connection region to form the double-chain BONT / A protein; the order of the HRV 3C enzyme cleavage and the enterokinase enzyme cleavage can be any of the following: 1) The HRV 3C enzyme cleavage and the enterokinase enzyme cleavage are carried out one by one; 2) The HRV 3C enzyme cleavage and the enterokinase enzyme cleavage are carried out simultaneously; the preferred method is that the HRV 3C enzyme cleavage and the enterokinase enzyme cleavage are carried out simultaneously; only two foreign amino acids GP remain at the N-terminus of the BONT / A light chain in the double-chain BONT / A obtained after further purification of the enzyme cleavage product. For reBONT / A-1 (i.e., Figure 1The recombinant protein expressed by the reBONT / A-1 gene can separate the HIS-GSTs tag and cleave the single-chain BONT / A light and heavy chain connection regions to form a double-chain BONT / A protein only by enterokinase digestion; the double-chain BONT / A protein sequence obtained after further purification of the digestion product is exactly the same as that of natural BONT / A, without any residual foreign amino acids. Referring to the double restriction enzyme site design of patent CN116440281A, after digestion, in addition to 2 foreign amino acids GP remaining at the N-terminus of the BONT / A light chain, 6 foreign amino acids LEVLFQ remain at the C-terminus of the BONT / A light chain, and 2 foreign amino acids GP remain at the N-terminus of the BONT / A heavy chain. Referring to the double restriction enzyme site design of patent CN118006523A, after digestion, in addition to 1 foreign amino acid S remaining at the N-terminus of the BONT / A light chain, 5 foreign amino acids LVPRG remain at the C-terminus of the BONT / A light chain, and 1 foreign amino acid S remains at the N-terminus of the BONT / A heavy chain.

[0058] The specific sequences of the components used in the above construction are shown in Table 1 below.

[0059] Table 1 Sequences used in the present invention

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Example 2 Transformation of the recombinant plasmid of the present invention into host cells

[0069] The recombinant plasmids containing reBONT / A and reBONT / A-1 constructed in Example 1 above were respectively transformed into host cells: an appropriate amount of the recombinant plasmid was gently mixed with Escherichia coli C41(DE3) competent cells and then incubated on ice for 30 min, heat shocked at 42 °C for 30 seconds, quickly placed on ice for 2 min, added with SOC medium, incubated at 37 °C and 220 rpm for 1 hour, and an appropriate amount of the revived bacterial solution was spread on an LB plate containing 50 μg / ml Kan and cultured overnight at 37 °C until obvious colonies grew.

[0070] Example 3 Amplification culture and induced expression of host cells of the present invention

[0071] Components and ratios of the culture medium: LB medium (10 g / L yeast peptone, 5 g / L yeast extract, 10 g / L sodium chloride), TB medium (12 g / L yeast peptone, 24 g / L yeast extract, 2.3 g / L potassium dihydrogen phosphate, 12.5 g / L dipotassium hydrogen phosphate, 4 ml / L glycerol)

[0072] Amplification culture: Pick monoclonal strains from the above transformation plate and inoculate them into LB medium, and shake culture at 37°C and 220 rpm until OD 600 reaches about 2.0, add a sterilized glycerol solution to mix into a bacterial suspension with a final glycerol concentration of 20%, aliquot into sterile cryotubes, and store at -80°C;

[0073] Induced expression: Inoculate the above glycerol bacteria into LB liquid medium at a ratio of 0.1%, and shake culture at 37°C and 220 rpm for 6 - 7 h until OD 600 reaches about 1.0, inoculate into TB liquid medium at a ratio of 2 - 3%, and shake culture at 37°C and 220 rpm for about 2 - 3 h until OD 600 reaches about 1.0, add IPTG with a final concentration of 0.5 mM to it for induction, and continue to shake culture at 25°C and 220 rpm overnight (about 16 h). After the induction is completed, sample to measure the OD of the culture 600 value, and collect the culture, centrifuge at 12000 rpm for 10 min to collect the bacterial cells. Induced OD 600 can be selected from 0.5 - 2, the IPTG concentration can be selected from 0.2 mM - 1 mM, the temperature can be selected from 20°C - 30°C, and the induction time can be selected from 5 h - 16 h.

[0074] Example 4 Detection of reBONT / A expression level

[0075] Use Wuhan Sanying GST Tag Monoclonal antibody (primary antibody) and HRP - conjugated Goat Anti - Mouse (secondary antibody) to perform Western Blot detection on bacteria under different induced expression conditions, and the results are as Figure 2 shown. Figure 2 It is the Wsetern blot result of the expression of reBONT / A fusion protein under different induction conditions, where Figure 2 a is the expression of reBONT / A fusion protein under different induction temperatures and inducer concentrations, Figure 2b shows the expression of the reBONT / A fusion protein under different induction durations. It can be seen that the reBONT / A of the present invention is expressed in the engineered bacteria under different induction temperatures, different inducer concentrations and different induction durations.

[0076] Example 5 Coarse purification of the reBONT / A fusion protein

[0077] a. Protein extraction: The bacterial cells collected in step 4 were resuspended at a ratio of 1:10 (W / V) using Lysis Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 20 mM imidazole), and then broken three times at a low temperature with a high-pressure homogenizer at a pressure of 700 - 800 bar. Then, the broken solution was centrifuged at 4°C and 15,000 g for 60 min to collect the supernatant.

[0078] b. Protein capture: The reBONT / A fusion protein in the supernatant was captured using an AKTA pure150 protein purifier and a NanoChrom NW Rose Ni FF chromatography column. The method is as follows: The chromatography column was rinsed with 5 column volumes of purified water, and then equilibrated with 5 column volumes of Lysis Buffer. Then, the broken supernatant was loaded at a certain flow rate to allow the fusion protein to fully bind to the chromatography packing material. After the loading was completed, the chromatography column was re-equilibrated with 20 column volumes of Lysis Buffer, and then linearly eluted with Elution Buffer (20 mM phosphate buffer + 300 mM sodium chloride + 300 mM imidazole) to obtain the reBONT / A fusion protein HIS-GSTs-BONT / A. The results were detected by SDS-PAGE experiment, as Figure 3 shown. Lane 2 is the reBONT / A fusion protein, and there is an obvious target band at about 175 kDa, which is the target protein of reBONT / A of the present invention obtained by coarse purification.

[0079] Example 6 Removal and purification of the tag protein

[0080] For the reBONT / A target protein obtained by coarse purification in Example 5, further protease digestion was carried out. The 3C protease (manufacturer: Novoprotein) was used to cut the 3C protease cleavage site between HIS-GSTs and BONT / A to obtain single-chain BONT / A. The results are as Figure 4 shown, and there is an obvious target band at about 150 kDa. The single-chain BONT / A was further digested with enterokinase (manufacturer: Novoprotein). Excitingly, the inventors first discovered that there is no need to introduce an additional protease cleavage site between the light chain and the heavy chain of single-chain BONT / A, and the light chain and the heavy chain can be effectively cleaved under the action of recombinant enterokinase. The results are as Figure 4 shown. Figure 4SDS-PAGE results of reBONT / A fusion protease digestion. Lane 2 is the reBONT / A fusion protein, lane 3 is the BONT / A single-chain protein and HIS-GSTs tag protein after digestion by 3C protease, lane 4 is the BONT / A double-chain protein and HIS-GSTs tag protein after digestion by enterokinase, and lane 5 is the result of double-chain dissociation of the sample in lane 4 under reducing conditions. It can be seen that the 150kd protein is cleaved by enterokinase into 100kDa (heavy chain) and 50kDa (light chain), and the cleaved light chain and heavy chain spontaneously form a dimer structure through disulfide bonds.

[0081] The digested sample was purified again using the NW Rose Ni FF chromatography column. The flow-through fraction after loading is the recombinant BONT / A with a dimer structure, while the cleaved tag protein HIS-GSTs, as well as the 3C protease and recombinant enterokinase carrying HIS tags, are all bound to the chromatography column. The results are as Figure 5 shown. Figure 5 SDS-PAGE results of the purification of the reBONT / A fusion protein of the present invention. Lane 2 is the purified BONT / A double-chain protein, and lane 3 is the HIS-GSTs tag protein. It can be seen that the purified flow-through fraction is the 150kd recombinant BONT / A, while the eluted fraction is mainly the heteroprotein with the 25kd HIS-GSTs tag protein.

[0082] The recombinant BONT / A sample in the flow-through fraction was purified by Nano-UniGel-30 DEAE chromatography as follows: The chromatography column was rinsed with 5 column volumes of purified water, and then equilibrated with 5 column volumes of DEAE equilibration buffer (20 mM Tris, pH 8.0). Then, the above-mentioned recombinant BONT / A sample in the flow-through fraction was loaded at a certain flow rate to allow the protein to fully bind to the chromatography packing material. After loading, the chromatography column was re-equilibrated with 5 column volumes of equilibration buffer, and then linearly eluted with DEAE elution buffer (20 mM Tris, 0.5 M sodium chloride, pH 8.0) to obtain the recombinant BONT / A protein. The results were detected by SDS-PAGE experiment, and the results are as Figure 6 shown. Figure 6 SDS-PAGE results of the purification of the BONT / A double-chain protein of the present invention. Lane 2 is the BONT / A double-chain protein with a purity greater than 97% after purification, and lane 3 is the verification result of double-chain dissociation under reducing conditions. It can be seen that the recombinant BONT / A with a protein purity greater than 97% is obtained in the present invention, and it is respectively the light chain and the heavy chain after reduction, indicating that the obtained recombinant BONT / A protein is a light chain and heavy chain dimer structure protein.

[0083] Example 7 Determination of the virulence of the recombinant double-chain BONT / A prepared by the present invention

[0084] The highly pure recombinant double-stranded BONT / A obtained in Example 6 using the reBONT / A of the present invention was intraperitoneally injected into Kunming mice, and its LD 50 was approximately 0.01 ng (see Table 2). At the same time, on the basis of reBONT / A of the present invention, referring to CN116440281A of the prior art patent document 1, an HRV 3C cleavage site was introduced between the light chain and heavy chain of BONT / A as a positive control strain for the molecular design of the traditional double protease cleavage site, and a highly pure recombinant double-stranded BONT / A sample was prepared. Under the same conditions, the LD 50 of the positive control BONT / A was approximately 0.05 ng. In comparison, the virulence of the recombinant BONT / A obtained in the present invention was approximately 5 times that of the positive control. It can be seen that in view of the reBONT / A designed in the present invention, the recombinant double-stranded BONT / A prepared has less residual foreign amino acids and stronger virulence. Similarly, for the reBONT / A-1 of the present invention, the highly pure recombinant double-stranded BONT / A obtained in Examples 4-6 above was used, and the virulence detection experiment also showed that its virulence was significantly higher than that of the above-mentioned positive control.

[0085] Table 2 Virulence determination of the recombinant double-stranded BONT / A of the present invention and the BONT / A with the traditional double cleavage site

[0086]

[0087]

[0088] The above examples of the present disclosure are merely examples for clearly explaining the present disclosure, and are not intended to limit the implementation manners of the present disclosure. For those of ordinary skill in the art, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to enumerate all the implementation manners here. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present disclosure shall be included within the protection scope of the claims of the present disclosure.

Claims

1. A recombinant botulinum neurotoxin type A (BONT / A) nucleic acid expression cassette, characterized in that, It sequentially includes the following elements from the 5'-end to the 3'-end: nucleic acid encoding a tag protein, nucleic acid encoding a cleavage site, BONT / A nucleic acid sequence; wherein, no exogenous protease recognition site is introduced between the light chain and the heavy chain of the BONT / A.

2. The nucleic acid expression cassette according to claim 1, wherein The tag protein includes one or more of HIS tag, GSTs tag, MBP tag, NusA tag, SUMO tag, TrxA tag; the cleavage site includes one or more of HRV 3C cleavage site, enterokinase cleavage site.

3. The nucleic acid expression cassette according to claim 1 or 2, wherein The nucleic acid expression cassette includes the following (1) or (2): (1) reBONT / A gene: It sequentially includes the following nucleic acid elements from the 5'-end to the 3'-end: HIS tag - GSTs tag - HRV 3C cleavage site - BONT / A; or (2) reBONT / A-1 gene: It sequentially includes the following nucleic acid elements from the 5'-end to the 3'-end: HIS tag - GSTs tag - Linker - enterokinase cleavage site - BONT / A.

4. The nucleic acid expression cassette according to any one of claims 1-3, characterized in that, The amino acid sequence of the HIS tag can be MGSS(H) n , where n is an integer from 6 to 10. Preferably, the nucleotide sequence of the HIS tag is as shown in SEQ ID NO.1, and the amino acid sequence of the HIS tag is as shown in SEQ ID NO.2; the nucleotide sequence of the GSTs tag is as shown in SEQ ID NO.3, and the amino acid sequence of the GSTs tag is as shown in SEQ ID NO.4; the nucleotide sequence of the HRV 3C cleavage site is as shown in SEQ ID NO.5, and the amino acid sequence of the HRV 3C cleavage site is as shown in SEQ ID NO.6; the amino acid sequence of the Linker can be (GGGGSGGGS) n , where n is an integer ≥ 1. Preferably, n = 1. The nucleotide sequence of the Linker is as shown in SEQ ID NO.7, and the amino acid sequence of the Linker is as shown in SEQ ID NO.8; the nucleotide sequence of the enterokinase cleavage site is as shown in SEQ ID NO.9, and the amino acid sequence of the enterokinase cleavage site is as shown in SEQ ID NO.10; the nucleotide sequence of the BONT / A is as shown in SEQ ID NO.11, and the amino acid sequence of the BONT / A is as shown in SEQ ID NO.

12.

5. The nucleic acid expression cassette according to claim 4, wherein The nucleotide sequence of the reBONT / A is as shown in SEQ ID NO.13, and the amino acid sequence of the reBONT / A is as shown in SEQ ID NO.14; the nucleotide sequence of the reBONT / A-1 is as shown in SEQ ID NO.15, and the amino acid sequence of the reBONT / A-1 is as shown in SEQ ID NO.

16.

6. A recombinant vector, characterized in that, It includes the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5; preferably, the recombinant vector is a pET series vector; more preferably, the pET series vector is pET-28a.

7. A recombinant expression bacterium, characterized in that, It includes the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5, or the recombinant vector according to claim 6; preferably, the recombinant expression bacterium is Escherichia coli; more preferably, the Escherichia coli includes E.coli BL21(DE3), E.coli Origami B(DE3), E.coli Rosetta Blue(DE3), E.coli C41(DE3); most preferably, the Escherichia coli is the E.coli C41(DE3) strain.

8. A recombinant botulinum neurotoxin type A (BONT / A) protein, characterized in that, It is encoded and expressed by the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5.

9. A method for constructing the recombinant expression bacterium, characterized in that, The recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5 is used to construct a recombinant expression plasmid by restriction enzyme digestion and ligation or one-step cloning, and the recombinant expression plasmid is transformed into Escherichia coli competent cells by heat shock method.

10. A method for preparing recombinant botulinum neurotoxin type A (BONT / A) expressed in the large intestine, characterized in that, It includes the steps of fermenting with the recombinant expression bacterium according to claim 7, digesting with enterokinase, cutting the connection region between the single-chain BONT / A light chain and heavy chain, and the cut light chain and heavy chain form a double-chain BONT / A protein, and purifying to obtain the recombinant BONT / A; preferably, preliminary purification by NTA affinity chromatography is also included before enterokinase digestion.

11. The preparation method according to claim 10, wherein The method includes obtaining a crude purified solution by subjecting the cells of the recombinant expression bacterium to homogenization disruption, centrifugation, and filtration; the crude purified solution is subjected to further purification, including any one or more of NTA affinity chromatography, GST affinity chromatography, protease digestion, and DEAE ion exchange chromatography; preferably, the further purification includes any one of the following: (1) NTA affinity chromatography - protease digestion - NTA affinity chromatography - DEAE ion exchange chromatography; or (2) NTA affinity chromatography - GST affinity chromatography - protease digestion - GST affinity chromatography - DEAE ion exchange chromatography; more preferably, the protease digestion includes any one or more of HRV 3C digestion or enterokinase digestion.

12. The preparation method according to claim 10 or 11, characterized in that, For the preparation of the recombinant protein expressed by the reBONT / A gene, HRV 3C enzyme is used to cleave the HRV 3C recognition site to separate the HIS-GSTs tag and the single-chain BONT / A protein, and enterokinase is used to cleave the BONT / A light chain and heavy chain connection region to form the double-chain BONT / A protein; the order of the HRV3C digestion and enterokinase digestion can be any one of the following: 1) HRV 3C digestion and enterokinase digestion are carried out one by one; 2) HRV 3C digestion and enterokinase digestion are carried out simultaneously; preferably, HRV 3C digestion and enterokinase digestion are carried out simultaneously; the double-chain BONT / A obtained after further purification of the digestion product has only two foreign amino acids remaining at the N-terminus of only the BONT / A light chain.

13. The preparation method according to claim 10 or 11, characterized in that, For the preparation of the recombinant protein expressed by the reBONT / A-1 gene, only enterokinase digestion is required to separate the HIS-GSTs tag and cleave the BONT / A light chain and heavy chain connection region to form the double-chain BONT / A protein; the double-chain BONT / A protein sequence obtained after further purification of the digestion product is completely identical to the natural BONT / A, without any foreign amino acid residues.

14. Use of the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5, the recombinant vector according to claim 6, the recombinant expression bacterium according to claim 7, the recombinant botulinum toxin type A (BONT / A) protein according to claim 8, the method for constructing the recombinant expression bacterium according to claim 9, and the preparation method of the recombinant botulinum toxin type A (BONT / A) expressed in Escherichia coli according to any one of claims 10-13 in the preparation of BONT / A.

15. Use of the recombinant botulinum toxin type A (BONT / A) nucleic acid expression cassette according to any one of claims 1-5, the recombinant vector according to claim 6, the recombinant expression bacterium according to claim 7, the recombinant botulinum toxin type A (BONT / A) protein according to claim 8, the method for constructing the recombinant expression bacterium according to claim 9, and the preparation method of the recombinant botulinum toxin type A (BONT / A) expressed in Escherichia coli according to any one of claims 10-13 in the medical and cosmetic fields; preferably, the use is a medical aesthetic product or a clinical drug.

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