High-efficiency thymosin alpha 1 expression gene and recombinant lactic acid bacteria

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a gene for efficiently expressing thymosin α1 (Tα1), recombinant lactic acid bacteria, and their applications. This invention provides a Tα1 encoding gene specifically optimized for *Lactococcus lactis* NZ9000, which significantly improves expression efficiency in the host through full-sequence codon adaptation. This gene was cloned into the vector pNZ8148-M, constructing the recombinant expression plasmid NZ9000 / pNZ8148-M. usp45 + T α1 The strain was successfully transformed into the host bacterium NZ9000, yielding recombinant lactic acid bacteria NZ9000 / pNZ8148-M. usp45 + Tα1 This recombinant strain can efficiently secrete the Tα1 fusion protein USP under Nisin induction. 45 +Tα1, the protein is digested in vitro by enterokinase to release active Tα1. The Tα1 produced by the method of this invention has high yield, high purity, high biological activity and excellent safety, providing a new technical platform for the low-cost, large-scale and safe production of Tα1.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a gene that efficiently expresses thymosin α1 and a recombinant lactic acid bacterium. Background Technology

[0002] Thymosin α1 (Tα1) is a small polypeptide composed of 28 amino acids with potent immunomodulatory activity, widely used clinically in the treatment of chronic hepatitis B, adjuvant cancer therapy, and immunodeficiency diseases. Currently, its production mainly relies on chemical synthesis, but this method involves complex purification processes and high costs, making it difficult to meet the demands of large-scale industrialization. As an alternative, heterologous expression using genetically engineered bacteria is an effective way to reduce costs. *Escherichia coli* is a commonly used prokaryotic expression host, but as an opportunistic pathogen containing endotoxins (lipopolysaccharide), it poses serious biosafety risks, limiting its application in the pharmaceutical field.

[0003] Lactic acid bacteria, especially *Lactococcus lactis*, are non-pathogenic, do not produce endotoxins, and can be taken orally directly, thus being recognized as generally safe (GRAS). *Lactococcus lactis* also possesses the ability to secrete and express exogenous proteins, making it an ideal host for expressing bioactive peptides. However, successfully applying lactic acid bacteria to the efficient production of Tα1 still faces significant technical challenges. First, codon bias is a core bottleneck: the human Tα1 gene is rich in rare codons found in lactic acid bacteria, leading to translational obstacles and extremely low expression levels, failing to meet industrial requirements. Second, secretion efficiency is low: even if expression is achieved, the target protein remains intracellularly, not only inhibiting host bacterial growth due to accumulation but also increasing the difficulty and cost of downstream cell disruption and purification; furthermore, the signal peptides used in existing secretion systems have poor compatibility with Tα1, resulting in a low proportion secreted extracellularly. Third, the controllability of the expression system and product activity also need to be considered: constitutive expression may exert continuous stress on the host, while an ideal induction system should be able to precisely control the expression timing to balance bacterial growth and product synthesis.

[0004] While existing technologies have explored solutions to the aforementioned problems, a breakthrough has not yet been achieved in unifying high yield, high secretion rate, high safety, and high activity. Therefore, there is an urgent need to develop an efficient expression platform to overcome the inherent limitations of existing technologies and provide a safe, efficient, low-cost preparation strategy for Tα1 suitable for large-scale production. Summary of the Invention

[0005] To address the aforementioned problems, a first aspect of the present invention provides a gene for efficiently expressing thymosin α1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] SEQ ID NO.1: AGTGAT GCT GCA GTT GAT ACA AGT TCA GAA ATT ACT ACT AAAGAT CTT AAA GAA AAG AAA GAA GTT GTT GAA GAA GCT GAA AAT.

[0007] SEQ ID NO.1 is a thymosin α1 encoding gene specifically designed for the efficient expression of thymosin α1 by *Lactococcus lactis* NZ9000. This sequence is not a natural gene, but rather an artificially synthesized sequence optimized based on the codon preferences of *Lactococcus lactis*. Its core design principle is to deeply modify the nucleotide sequence without altering the final encoded amino acid sequence (i.e., ensuring the product is completely identical to natural human thymosin α1). This optimized sequence has significant advantages: First, it completely eliminates rare codons from lactic acid bacteria and replaces them all with high-frequency synonymous codons preferred by the host, thus eliminating "traffic jams" in the translation process and greatly improving the protein synthesis rate. Second, by adjusting the overall GC content of the gene to match the average level of the *Lactococcus lactis* genome (approximately 38%), it effectively avoids the formation of secondary structures in the mRNA that hinder translation, ensuring the accessibility and stability of the mRNA. Furthermore, the sequence design avoids unstable elements such as repetitive sequences and introduces restriction enzyme sites that facilitate cloning. Through optimization measures, SEQ ID NO.1 can achieve high-level and high-efficiency stable expression within the lactic acid bacteria host.

[0008] In a second aspect, the present invention provides a recombinant expression plasmid comprising the gene described in the first aspect.

[0009] As a preferred technical solution, the recombinant expression plasmid is pNZ8148-M: USP45 + Tα1 Using the laboratory-constructed high-secretion expression vector pNZ8148-M (patent pending, application number: 202411244441.6, the contents of which are incorporated herein by reference) as a backbone, the encoding gene of the lactic acid bacteria exocrine protein USP45 was fused with the encoding gene of thymosin α1. Thymosin α1 was successfully transported extracellularly via USP45, effectively solving the technical challenge of transmembrane transport of short intracellular peptides. Simultaneously, due to the presence of the fusion protein, thymosin α1 is not easily digested by intracellular enzymes, ensuring its stability. Furthermore, an enterokinase site was added between the fusion protein USP45 and Tα1, allowing it to dissociate and release thymosin α1 in the intestine, thus exerting its biological activity.

[0010] In a third aspect, the present invention provides a recombinant lactic acid bacteria comprising the recombinant expression plasmid described in the second aspect.

[0011] As a preferred technical implementation, the recombinant lactic acid bacteria described in this scheme is NZ9000 / pNZ8148-M:usp45 + Tα1. The chassis strain is selected as NZ9000, a derivative of *Lactococcus lactis*. This strain naturally lacks the structural gene (nisA) encoding nisin in its genome, but retains the complete Nisin sensing and regulatory system (NisRK). This unique genetic characteristic prevents it from synthesizing Nisin autonomously, but it can exhibit a highly sensitive response to exogenously added Nisin. Based on this, this scheme precisely induces the expression of the usp45 + Tα1 fusion protein in NZ9000 / pNZ8148-M by exogenously adding Nisin.

[0012] Using NZ9000 as a recombinant lactic acid bacteria has multiple advantages: Excellent biosafety: This strain has been recognized as a "generally considered safe" (GRAS) strain by the US FDA and the European Food Safety Authority, and has a wide range of applications in the field of food fermentation, possessing extremely high biosafety.

[0013] The expression system is mature and efficient: it has a clear genetic background, mature molecular manipulation techniques, and high compatibility with the pNZ8148-M vector, which can ensure that the exogenous gene is expressed efficiently and controllably under Nisin induction.

[0014] High product stability and yield: NZ9000 has low protease activity, which can effectively reduce the degradation of active peptides secreted into the extracellular space, thereby significantly improving the stability and final yield of the target product.

[0015] At the application level, this technology fundamentally optimizes the production process by constructing recombinant plasmids capable of secreting and expressing thymosin α1. It eliminates the expensive and cumbersome downstream processing steps of traditional methods, such as cell disruption, separation, and purification, and allows the engineered bacteria to be directly prepared into "live drugs" or functional formulations that can be taken orally or used as additives. Once this formulation enters the intestines, the secreted fusion protein releases bioactive thymosin α1 under the action of intestinal enzymes. This strategy simplifies the production process, reduces preparation costs, and has high application value.

[0016] In a fourth aspect, the present invention provides a method for efficiently expressing thymosin α1, the method employing the recombinant lactic acid bacteria of the third aspect.

[0017] As a preferred technical solution, the method for efficiently expressing thymosin α1 uses a temperature of 26℃, a Nisin concentration of 1.5 ng / mL, and an induction time of 10 hours.

[0018] In a fifth aspect, the present invention provides a method for preparing thymosin α1, the method comprising the induction method described in the fourth aspect.

[0019] In a sixth aspect, the present invention provides a thymosin α1, which is prepared by the method of the fifth aspect.

[0020] A seventh aspect of the present invention provides the use of the thymosin α1 described in the sixth aspect in the preparation of immunomodulatory drugs or health products.

[0021] Through the above technical solutions, the present invention achieves the following technical effects: (1) Achieved efficient and controllable expression of thymosin α1 in lactic acid bacteria: Through whole-sequence codon optimization, the core bottleneck of codon preference mismatch between human genes and the Lactococcus lactis host was solved, greatly improving translation efficiency and mRNA stability. Combined with precise regulation by the Nisin inducible promoter, the target protein can be efficiently induced to express only after the bacteria have grown to the optimal state, effectively avoiding the potential toxicity of exogenous proteins to host cells.

[0022] (2) High-proportion, high-activity extracellular secretion was achieved: Using the USP45 signal peptide inherent in the pNZ8148-M vector, the expression product was successfully secreted into the extracellular culture medium. This not only greatly simplified the downstream purification process (no cell disruption required), but more importantly, it ensured that the fusion protein USP45+Tα1 could release a large amount of thymosin α1 in the intestine, avoiding the formation of inclusion bodies of the target protein in the bacteria, thereby obtaining the target product with complete natural structure and high biological activity.

[0023] (3) A safe, stable, and high-yield production platform was constructed: Lactococcus lactis NZ9000, a GRAS-grade, endotoxin-free, and low-protease-activity strain, was selected as the host, which fundamentally ensured the biosafety of the product and reduced the degradation of secreted products. The recombinant system has a clear genetic background and mature operation. After condition optimization, the secretion expression level and stability of thymosin α1 were significantly improved, fully meeting the needs of large-scale industrial production.

[0024] (4) Verification of the product's excellent biological function: Following national standards, an E-rosette experiment was performed on the enzyme-digested thymosin Tα1. The results showed that the rosette binding rate of the enzyme-digested product was significantly increased compared to the control group, confirming the biological activity of thymosin Tα1. Furthermore, in vitro activity detection by flow cytometry confirmed that the recombinant thymosin α1 prepared in this invention can effectively promote T cell differentiation and maturation and remodel CD4+. + / CD8 +It achieves subpopulation balance and exerts a broad regulatory effect on immune cells such as B cells and NK cells. Its immunomodulatory activity is comparable to that of commercial products, which fully demonstrates the feasibility of this technical route. Attached Figure Description

[0025] Figure 1 : USP45 + Tα1 Electrophoresis diagram of the fusion gene; lane M is Marker DL2000; lane 1 is the amplification result using primers EUSPF3 and TP1, product size 1343 bp; lane 2 is the amplification result using primers EUSPF3 and TP2, product size 1378 bp; lane 3 is the amplification result using primers EUSPF3 and TP3, product size 1410 bp; lane 4 is the amplification result using primers EUSPF3 and TP4, product size 1435 bp.

[0026] Figure 2 Recombinant plasmid pNZ8148-M: USP45 + Tα1 PCR identification results: Lane M is Marker DL5000; Lane 1 is plasmid pNZ8148, product size 987 bp; Lane 2 is recombinant plasmid pNZ8148-M: usp45 + Tα1, product size 2476 bp.

[0027] Figure 3 Recombinant plasmid pNZ8148-M: USP45 + Tα1 Double enzyme digestion identification results: Lane M is Marker DL5000; Lane 1 is recombinant plasmid pNZ8148-M:usp45+Tα1; Lane 2 is recombinant plasmid pNZ8148-M:usp45+Tα1 double digestion.

[0028] Figure 4 Western blot identification results of induced expression of fusion protein USP45+Tα1; Lane M is the protein marker; Lane 1 is the concentrated sample of NZ9000pNZ8148-M:usp45+Tα1 culture medium after Nisin induction; Lane 2 is the concentrated sample of NZ9000pNZ8148-M:usp45+Tα1 culture medium before induction; Lane 3 is the concentrated sample of NZ9000pNZ8148-M culture medium without the vector.

[0029] Figure 5Electrophoretic identification results of the fusion protein USP45+Tα1 after acetone concentration optimization; Lane M is the protein marker; Lane 1 is 46% acetone precipitation; Lane 2 is 48% acetone precipitation; Lane 3 is 50% acetone precipitation; Lane 4 is 50% acetone precipitation; Lane 5 is 52% acetone precipitation; Lane 6 is 54% acetone precipitation.

[0030] Figure 6 USP45+Tα1 expression levels at different Nisin working concentrations; lane M is the protein marker; lane 1 is the Nisin concentration (1 ng / mL); lane 2 is the Nisin concentration (1.5 ng / mL); lane 3 is the Nisin concentration (2 ng / mL); lane 4 is the Nisin concentration (2.5 ng / mL); lane 5 is the Nisin concentration (5 ng / mL); lane 6 is the Nisin concentration (10 ng / mL); lane 7 is the Nisin concentration (20 ng / mL); lane 8 is the Nisin concentration (30 ng / mL); lane 9 is the Nisin concentration (40 ng / mL).

[0031] Figure 7 USP45+Tα1 expression levels after induction at different temperatures; lane M is the protein marker; lane 1 is the induction temperature (20℃); lane 2 is the induction temperature (26℃); lane 3 is the induction temperature (30℃); lane 4 is the induction temperature (37℃); lane 5 is the induction temperature (40℃).

[0032] Figure 8 USP45+Tα1 expression levels after different induction times; lane M is the protein marker; lane 1 is the induction time (6h); lane 2 is the induction time (8h); lane 3 is the induction time (10h); lane 4 is the induction time (12h); lane 5 is the induction time (14h).

[0033] Figure 9 Western blot identification of the fusion protein USP45+Tα1 enterokinase before and after digestion; lane M is the protein marker; lane 1 is before digestion; lane 2 is after digestion for 24 h; lane 3 is after digestion for 48 h.

[0034] Figure 10 Group 1: Flow cytometry results.

[0035] Figure 11 Group 2: Flow cytometry results.

[0036] Figure 12 Group 3: Flow cytometry results.

[0037] Figure 13 Group 4: Flow cytometry results. Detailed Implementation

[0038] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the applicant provides an illustrative analysis through embodiments.

[0039] Unless otherwise stated, all percentages in this article refer to mass percentages.

[0040] Example 1: Recombinant expression plasmid pNZ8148-M: USP45 + Tα1 Construction 1. Gene optimization Based on the codon preference of *Lactococcus lactis* NZ9000, the full sequence of the coding gene for wild-type thymosin α1 (Tα1) was optimized by removing rare codons and adjusting the GC content. First, primers were designed to amplify the USP45 coding gene in *Lactococcus lactis*. Then, using four-overlap extension PCR methods, the nucleotide sequences encoding enterokinase-recognized cleavage peptides and thymosin α1 were sequentially ligated to the 3' end of the USP45 gene. A Kpn1 restriction site was added to the 5' end of this sequence, and an Xba I restriction site was added to the 3' end. The optimized sequence is shown in SEQ ID NO.1.

[0041] SEQ ID NO.1: AGT GAT GCT GCA GTT GAT ACA AGT TCA GAA ATT ACT ACT AAAGAT CTT AAA GAA AAG AAA GAA GTT GTT GAA GAA GCT GAA AAT The specific optimization process is as follows: (1) Determine the codon preference of the host lactic acid bacteria: Lactococcus lactis NZ9000 was selected as the host strain. The frequency of codon usage of the two host strains was queried by the bioinformatics analysis tool (Codon Usage Database). Preferred codons with a usage frequency ≥30% were screened out, and rare codons with a usage frequency <15% were removed.

[0042] (2) Codon optimization principle: Under the premise of not changing the amino acid sequence of thymosin α1 (to ensure its biological activity), codon optimization is performed on wild-type genes: all rare codons that are used less frequently in lactic acid bacteria are replaced with their preferred synonyms; at the same time, the overall GC content of the gene is adjusted to be close to the GC content of the host Lactococcus lactis NZ9000 genome (about 38%), so as to prevent the formation of secondary structures that affect transcription and translation due to excessive differences in GC content; in addition, the start codon (ATG) and stop codon (TAA) with the highest host recognition efficiency are selected, and continuous repetitive sequences or hairpin structures are avoided, thereby comprehensively improving the expression stability and translation efficiency of the gene in lactic acid bacteria.

[0043] (3) PCR extension amplification: EUSP3 and EUSPR primers were designed, and the USP45 encoding gene was amplified using Lactococcus lactis NZ9000 bacterial culture as a template. The PCR product was recovered and used as a template for the first overlapping extension PCR using EUSP3 and TP1 primers. The PCR product was then recovered and used as a template for the second overlapping extension PCR using EUSP3 and TP2 primers. Four overlapping extension PCRs were performed sequentially. The nucleotide sequences encoding enterokinase-recognized cleaved peptide chains and thymosin α1 were sequentially ligated to the 3' end of the USP45 gene. A Kpn1 restriction site was added to the 5' end of this sequence, and an Xba I restriction site was added to the 3' end. The electrophoresis results of the PCR extension amplification of this sequence are shown in [Figure number missing]. Figure 1 .

[0044] 2. Double enzyme digestion and ligation A lactic acid bacteria secretory expression vector was selected as the backbone vector. The Tα1 gene fragment obtained from PCR extension amplification was double-digested with restriction endonucleases Kpn1 and Xba I, respectively. The digestion products were separated by 0.8% agarose gel electrophoresis and recovered using a gel extraction kit. The vector size was 3165 bp, and the PCR extension product was 1435 bp. The recovered vector and gene fragment were mixed at a 1:3 molar ratio, and T4 DNA ligase was added. Ligation was carried out overnight at 16°C. The specific steps are as follows: (1) Selection of backbone vector: pNZ8148-M is a highly secretory expression vector modified from pNZ8148 (patent pending, application number: 202411244441.6, the contents of which are incorporated herein by reference). This vector has the following advantages: it is a food-grade vector with no risk of antibiotic resistance gene residue (containing only low-toxicity resistance genes); it has a strong lactic acid bacteria-specific promoter and terminator, which can efficiently regulate the transcription and translation of the target gene; it contains a secretion signal peptide coding sequence, which can guide the secretion of the target protein into the fermentation supernatant; the vector has a small molecular weight, is stable in replication within lactic acid bacteria, and is not easily lost. The pNZ8148-M vector is a Nisin-inducible expression vector containing the Nisin-inducible promoter PnisA, which can regulate the expression of the target gene by adding Nisin inducer. The expression regulation is flexible and suitable for large-scale fermentation production.

[0045] (2) Double digestion of vector and gene: pNZ8148-M backbone vector and fusion gene obtained by overlapping extension were extracted. USP45 + Tα1 The sample was double-digested using restriction endonucleases Kpn1 and Xba I. The digestion system (50 µL) consisted of 5 µL of 10×TangoBuffer, 1 µL of Kpn1, 1 µL of Xba I, and 43 µL of plasmid / PCR product. The digestion conditions were incubation at 37°C for 3 h. After incubation, 5 µL of 0.5 M EDTA solution was added to terminate the digestion reaction.

[0046] (3) Recovery and purification of enzyme digestion products: The reaction solution after terminating enzyme digestion was added to a 0.8% agarose gel (containing 0.5 μg / mL EB staining agent), and electrophoresis was performed at a constant voltage of 120V for 30 min using 1×TAE buffer to separate the digested vector backbone fragments and... USP45 + Tα1 Gene fragment isolation; observation of electrophoresis results using a gel imaging system to confirm complete enzyme digestion; cutting off the agarose gel block containing the target band with a clean scalpel, and recovering the vector backbone fragment (pNZ8148-M vector fragment) and... USP45 + Tα1 Gene fragments were recovered; after recovery, the concentration and purity of the recovered product were determined using a nucleic acid quantification instrument to ensure that the concentration of the recovered product was ≥50 ng / μL and OD0.05 260 / OD 280 The ratio is between 1.8 and 2.0, and the purity meets the requirements for subsequent ligation reactions.

[0047] (4) Connection reaction: The recovered USP45 +Tα1 The fragment was ligated to the linearized pNZ8148-M vector overnight at 4°C using T4 DNA ligase. Ligation system: 2 µL 10× ligation buffer, 1 µL digested plasmid, 5 µL digested PCR product, 1 µL ligase, and ddH2O to a final volume of 20 µL.

[0048] 3. Transformation The ligation product was transformed into *E. coli* MC1061 competent cells, plated on LB agar plates containing chloramphenicol (10 μg / mL), and incubated at 37°C for 16–18 h. The specific steps are as follows: Add 10 μL of the ligation product to 50 μL of E. coli MC1061 competent cells and incubate on ice for 30 min; heat shock at 42℃ for 90 s, then immediately incubate on ice for 20 min; add 900 μL of freshly prepared SOC liquid medium and culture at 37℃ and 160 rpm for 1 h with shaking; take 200 μL of the bacterial culture and spread it on LB solid medium containing 10 μg / mL chloramphenicol, and incubate at 37℃ for 16-18 h.

[0049] 4. Identification of recombinant plasmids PCR identification: Single colonies were picked and inoculated into LB broth containing 10 μg / mL chloramphenicol, and cultured at 37℃ with shaking at 220 rpm for 16-18 h. Using the cultured bacterial culture as a template, PCR amplification was performed using detection primers pN8F and pN8R (these primers are located outside the multiple cloning site of the vector pNZ8148-M). The PCR reaction system was 25 μL: 1 μL bacterial culture, 1 μL upstream primer, 1 μL downstream primer, 12.5 μL 2×Taq PCR Master Mix, and 9.5 μL sterile deionized water. PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 2 min, for a total of 30 cycles; 72℃ final extension for 10 min; 16℃ for 30 min. After PCR amplification, 0.8% agarose gel electrophoresis was performed for detection. The results are shown in the figure below. Figure 2 As shown in the figure, the positive bacteria amplified a target band between 2000 bp and 3000 bp, which is consistent with the theoretical fragment length (2486 bp).

[0050] Double enzyme digestion identification: Plasmids were extracted from PCR-amplified positive bacteria according to the instructions of the plasmid extraction kit and then subjected to double enzyme digestion identification. The enzyme digestion system (20 μL) was as follows: 10× buffer, 2 μL; KpnI, 1 μL; Xba I, 1 μL; plasmid, 17 μL. Digestion was performed at 37℃ for 1 h. The original plasmid and enzyme digestion products were detected by 0.8% agarose gel electrophoresis, and the results are shown in the figure. Figure 3As shown in the figure, a target band appeared around 1000 bp in the enzyme digestion product, which is basically consistent with the size of the inserted fragment (1235 bp), indicating that the fusion gene... USP45 + Tα1 It has been successfully ligated into the pNZ8148-M vector.

[0051] Sequencing confirmation: Using a positive recombinant plasmid as a template, the target fragment was amplified according to the method described in PCR identification, purified, and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. Analysis of the results using DNAstart software showed that pNZ8148-M: USP45 + Tα1 Successfully built.

[0052] Example 2: Recombinant lactic acid bacteria strain NZ9000 / pNZ8148-M: USP45 + Tα1 Preparation 1. Preparation method The successfully constructed recombinant expression plasmid was introduced into competent host lactic acid bacteria cells using electroporation, and positive recombinant lactic acid bacteria strains were obtained through screening. The specific steps are as follows: (1) Preparation of competent cells of host lactic acid bacteria: Lactococcus lactis NZ9000 was selected as the host strain to prepare competent cells. The specific steps were as follows: Lactococcus lactis was inoculated into 5 mL of M17 liquid medium (containing 0.5% glucose) and cultured statically at 30℃ for 16 h to obtain seed culture; the seed culture was inoculated into 20 mL of fresh M17 liquid medium (containing 0.5% glucose) at a ratio of 1:100 and cultured statically at 30℃ until OD 600 =Approximately 0.5. Pre-cool the bacterial suspension in an ice bath for 30 min, centrifuge at 4°C and 6500 rpm for 15 min, and collect the bacterial cells. Wash the bacterial cells five times with pre-cooled 10% sterile glycerol solution, centrifuging each suspension at 4°C and 6500 rpm for 15 min, and collect the bacterial cells each time. Finally, resuspend the bacterial cells in 200 μL of 10% sterile glycerol solution.

[0053] (2) Electroporation: Take 25 μL of NZ9000 competent lactic acid bacteria cells and 1-2 μg of recombinant expression plasmid (pNZ8148-M: USP45 + Tα1Gently mix the contents in a 1.5 mL EP tube and incubate on ice for 20 min. Transfer the mixture to a pre-chilled 0.1 cm electroporation cuvette and place the cuvette in the electroporator. Set the electroporation parameters: voltage 2.5 kV, capacitance 25 μF, resistance 50 Ω, and start electroporation. After electroporation, immediately add 900 μL of M17 medium (containing 0.5% glucose), gently mix by pipetting, and transfer to a 1.5 mL centrifuge tube. Incubate statically at 30 °C for 2 h, then centrifuge the bacterial culture at 6500 rpm for 5 min at room temperature. Discard 700 μL of supernatant. Mix the remaining supernatant with the bacterial cells and spread it onto an M17 solid plate containing 10 μg / mL chloramphenicol. Incubate at 30 °C for 24–48 h.

[0054] 2. Screening and identification of positive recombinant strains Pick 3-5 single colonies and inoculate them into M17 liquid medium containing 5 μg / mL chloramphenicol. Incubate at 30℃ for 16-18 h to obtain bacterial suspensions. Perform colony PCR identification on the bacterial suspensions using the same method as for recombinant plasmid identification. The identified positive strain is the genetically engineered bacterium NZ9000 / pNZ8148-M:usp45+Tα1.

[0055] Example 3: NZ9000 / pNZ8148-M: USP45 + Tα1 Induced expression 1. Seed culture: Remove the positive recombinant lactic acid bacteria strain from the -80℃ freezer and thaw it in an ice bath. Inoculate 200 μL of the bacterial culture into 10 mL of M17 liquid medium (containing 10 μg / mL chloramphenicol and 0.5% glucose by mass) and incubate statically at 30℃ for 16-18 h to obtain the primary seed culture. Inoculate the primary seed culture into 100 mL of fresh M17 liquid medium (containing 0.5% glucose by mass) at a volume ratio of 2% and incubate statically at 30℃ for 16-18 h to obtain the secondary seed culture.

[0056] 2. Expanded Culture and Induced Expression: The secondary seed culture was inoculated into 1000 mL of fresh M17 liquid medium (containing 0.5% glucose by mass) at a volume ratio of 2%, and cultured statically at 30°C until OD500. 600 =Approximately 0.6; add Nisin inducer to a final concentration of 1.5 ng / mL; adjust the culture temperature to 26℃ and culture statically for 8 hours.

[0057] 3. Fermentation broth treatment and supernatant collection: Transfer the fermentation broth to centrifuge tubes and centrifuge at 12,000 rpm and 4°C for 10 min. Collect the supernatant. Filter the supernatant through a 0.22 μm sterile filter membrane to obtain the pretreated fermentation broth. Place the fermentation broth in a dialysis bag and concentrate it 20 times using PEG6000 at 4°C.

[0058] 4. Identification of the fusion protein USP45+Tα1: The protein in the concentrated fermentation broth was separated by SDS-PAGE and detected by Western blot using a 6×His antibody. The specific steps of the Western blot detection are as follows: Proteins obtained from SDS-PAGE electrophoresis were transferred to a PVDF membrane using a transfer apparatus (transfer conditions: constant current 400mA, transfer for 40 min). After transfer, the PVDF membrane was placed in 5% skim milk blocking buffer and incubated at room temperature with shaking for 2 h to block non-specific binding sites. The blocking buffer was discarded, and the PVDF membrane was washed 5 times with TBST buffer (PBS buffer containing 0.1% Tween-20, pH 7.4), 6 min each time. His-Tag monoclonal antibody primary antibody was added and incubated at 4℃ for 10 h with shaking (1:1000 dilution) for overnight with shaking. The primary antibody was discarded, and the PVDF membrane was washed 5 times with TBST buffer, 6 min each time. HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution) was added and incubated at room temperature with shaking for 2 h. The secondary antibody was discarded, and the PVDF membrane was washed 5 times with TBST buffer, 6 min each time. The PVDF membrane was placed in ECL chemiluminescence reagent and incubated in the dark for 5 min, then exposed and imaged using a chemiluminescence imaging system.

[0059] The Western blot results of the fusion protein USP45+Tα1 are shown below. Figure 4 The presence of the target band near 72 kDa indicates that the constructed genetically engineered bacteria successfully secreted the fusion protein USP45+Tα1.

[0060] Example 4: Optimization of Acetone Concentration Take 10 mL of the concentrated solution prepared in Example 3, and add 5 times the volume of ice-cold acetone of different concentrations (46%, 48%, 50%, 52%, and 54%, respectively). Incubate overnight at 4°C. Then centrifuge at 12000 rpm for 30 min at 4°C, collect the precipitate, and place it in a ventilated area for 10 min to remove residual acetone. Resuspend the precipitate in 100 µL of pre-cooled PBS. Take 20 µL of each solution treated with different concentrations of acetone for SDS-PAGE electrophoresis. Figure 5 As shown, 50% acetone precipitation yielded the best results and the highest yield of fusion proteins.

[0061] The content of the fusion protein USP45+Tα1 was determined using the BCA protein quantification kit, following the kit's instructions. The specific steps were as follows: a standard protein curve was prepared; the USP45+Tα1 solution was appropriately diluted, BCA reagent was added, and the mixture was incubated at 37°C for 30 min. The absorbance (OD value) at 562 nm was measured using a microplate reader. The concentration of USP45+Tα1 in the solution was calculated based on the standard curve. The expression level of the fusion protein USP45+Tα1 was estimated based on the amount of fermentation broth and the concentration factor.

[0062] Example 5: Optimization of Induction Conditions The induction conditions were optimized using the method described in Example 3.

[0063] 1. Nisin working concentration optimization: Recombinant lactic acid overnight culture medium was passaged into nine 200mL M17 liquid culture media at a volume ratio of 2%, and incubated statically at 30℃. When OD 600 When the concentration reached 0.6, Nisin was added to achieve working concentrations of 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, and 40 ng / mL, respectively. The mixture was then incubated at 30°C for 8 hours. The fermentation broth was treated according to the method in Example 3, and the fusion protein USP45+Tα1 was precipitated with 50% acetone (v / v) according to the method in Example 4. All samples were separated by SDS-PAGE electrophoresis and identified by Western blot; the results are shown in [Figure 1]. Figure 6 As shown in the figure, the lactic acid engineered bacteria fusion protein secretion was maximized when induced statically with 1.5 ng / mL Nisin for 8 h.

[0064] 2. Temperature optimization: The recombinant lactic acid overnight culture medium was passaged at a volume ratio of 2% into nine 200 mL M17 liquid culture media and incubated statically at 30°C. When OD... 600 When the concentration reached 0.6, Nisin (working concentration 1.5 ng / mL) was added, and the samples were incubated at 20℃, 26℃, 30℃, 37℃, and 40℃ for 8 h, respectively. The fermentation broth was treated according to the method in Example 3, and the fusion protein USP45+Tα1 was precipitated with 50% acetone (v / v) according to the method in Example 4. Samples induced at different temperatures were separated by SDS-PAGE electrophoresis and identified by Western blot. The results are shown in [Figure 1]. Figure 7 As shown in the figure, the lactic acid engineered bacteria fusion protein secretion was the largest after static incubation at 26℃ for 8 hours.

[0065] 3. Optimization of induction time: Recombinant lactic acid overnight culture medium was passaged at a volume ratio of 2% into nine 200 mL M17 liquid culture media, and incubated statically at 30°C. When OD...600 When the concentration reached 0.6, 1.5 ng / mL Nisin was added, and the mixture was induced at 26°C for 6 h, 8 h, 10 h, 12 h, and 14 h, respectively. The fermentation broth was treated according to the method in Example 3, and the fusion protein USP45+Tα1 was precipitated with 50% acetone (v / v) according to the method in Example 4. All samples were separated by SDS-PAGE electrophoresis and identified by Western blot. The results are shown in [Figure 1]. Figure 8 As shown in the figure, the lactic acid engineered bacteria fusion protein secretion was maximized when the Nisin concentration was 1.5 ng / mL, the static culture temperature was 26℃, and the culture time was 10 h.

[0066] Through the above optimization, the concentration of USP45+Tα1 in the purified sample was 359.4 mg / mL, from which the optimal secretion amount of the fusion protein was calculated to be 17.97 mg / mL.

[0067] Example 1: Enterokinase in vitro cleavage of fusion protein USP45+Tα1 According to the enterokinase instructions, 50 μL of the fusion protein solution was mixed with 1 μL of enterokinase, and incubated at 4°C for 24 h and 48 h, respectively. The digested fusion protein solution and the undigested fusion protein were subjected to SDS-PAGE and Western blot analysis. The results are shown in the table below. Figure 9 As shown in the figure, the undigested fusion protein exhibits a specific band around 70 kDa, while the enterokinase-digested sample shows a band between 25 kDa and 35 kDa, which is the exocrine USP45. Because thymosin Tα1 has a small molecular weight of approximately 3 kDa and does not carry a 6×His tag after cleavage from the fusion protein, it cannot be detected by Western blot. These results indicate that enterokinase can effectively cleave the fusion protein, releasing thymosin. Based on the molecular weights of USP45 and thymosin Tα1, the fusion protein USP45+Tα1 secreted by the genetically engineered bacteria is predicted to be a dimer.

[0068] Example 2: Detection of Thymosin Tα1 activity 1. E Rose Garland Experiment (1) Experimental materials Test sample: The fusion protein USP45+Tα1 digested with enterokinase was diluted with Hank's solution to 50 μg / mL, 100 μg / mL and 200 μg / mL for later use.

[0069] Positive control: Thymosin α1 for injection (Gitai®), diluted with Hank's solution to 200 μg / mL and 100 μg / mL.

[0070] Negative / blank control: Hank's solution (containing 10% inactivated fetal serum absorbed by SRBC).

[0071] T-cell source: Bikmann heparin sodium anticoagulated pig blood (to be used within 2 weeks after blood collection).

[0072] Sheep red blood cells: Bickman defibrinated sheep blood, used within 2 weeks after blood collection, and prepared into a 5% suspension after washing 3 times with Hank's solution.

[0073] Lymphocyte separation medium: Biosharp human peripheral blood lymphocyte separation medium (specific gravity 1.077), stored at room temperature.

[0074] (2) Experimental methods T lymphocyte isolation: Take 2 mL of heparin sodium anticoagulated porcine blood and dilute it with an equal volume of Hank's solution. Take a 15 mL centrifuge tube, add 4 mL of lymphocyte separation solution, and slowly add 4 mL of diluted blood along the tube wall, keeping the interface clear. Centrifuge horizontally at 450×g for 30 min at room temperature. Aspirate the grayish-white cloud layer (lymphocyte layer), transfer it to a new centrifuge tube, add 10 mL of Hank's solution, centrifuge at 250×g for 10 min, wash, and repeat twice. Stain with trypan blue and count the cells; cells with ≥95% viability are acceptable for use.

[0075] E receptor desensitization treatment: Prepare a suspension of lymphocytes (approximately 2 × 10⁻⁶ cells). 6 (cells / mL) were placed in a 45°C water bath and heated for 35 min (shaking every 5 min). After heating, pre-warmed Hank's solution at 37°C was added, and the cells were washed by centrifugation at 250×g for 10 min, repeated twice. The cell viability after E removal treatment should be ≥85%.

[0076] E-rosette assay: De-E treated T cells were adjusted to 2 × 10⁶ cells / cells using Hank's solution containing 10% inactivated fetal bovine serum. 6 cells / mL. Samples were added according to the groups in Table 1 (total volume 400 μL per tube).

[0077] Table 1 Grouping After adding the sample, mix gently and incubate in a 37°C water bath for 2 hours. After incubation, add 5% SRBC suspension (4×10⁻⁶). 7(Cells / mL, mix thoroughly before use), centrifuge at 800×g for 5 min after gentle mixing, then transfer to a 4℃ refrigerator and incubate overnight (16 h-20 h). The next day, discard the supernatant, add 100 μL of 0.8% glutaraldehyde along the tube wall, gently vortex to resuspend, and fix at 4℃ for 15 min. Prepare cell suspension slides, stain with Wright-Giemsa, and count 200 lymphocytes under a high-power microscope (×400). Record the number of positive cells binding ≥3 SRBCs. Count twice per tube and take the average value. Two parallel tubes are used for each group.

[0078] (3) Experimental results The experimental results are shown in the table below. The differences in E-rosette rates between the 200 μg / mL, 100 μg / mL, and 50 μg / mL purified thymosin groups and the blank control were 10.75%, 11.62%, and 10%, respectively, all of which were not lower than the national standard for the determination of thymosin activity (National Drug Standard), confirming that the expressed thymosin has biological activity.

[0079] Table 2 Experimental Results 2. Flow cytometry detection of thymosin bioactivity (1) Experimental grouping and treatment The whole blood was divided into the following 4 groups: Group 1 (blank control group): Whole blood samples, without any processing, were used directly for subsequent testing.

[0080] Group 2 (E receptor removal control group): Whole blood samples were incubated in a 45°C water bath for 30 min to remove E receptors (sheep erythrocyte receptors, i.e., CD2 molecules, mainly used to eliminate T cells or other related cell functions) from the cell surface. An equal volume of PBS was added after treatment as a negative control.

[0081] Group 3 (thymopeptide group): Whole blood samples were first treated in a 45℃ water bath for 30 min to remove E receptors, and then thymopeptides (final concentration 20 μg / mL) were added after expression, purification and enzyme digestion.

[0082] Group 4 (thymopeptide drug group): Whole blood samples were first treated in a 45°C water bath for 30 minutes to remove E receptors, and then thymopeptide drug preparation (Jitai-thymosin for injection, concentration 20 μg / mL), batch number H20030407 was added.

[0083] (2) Test results In vitro bioactivity verification results show that the recombinant thymosin α1 prepared in this invention has clear and effective immunomodulatory activity. The detection results are shown in [see attached table]. Figure 10-13 .

[0084] A comprehensive analysis of multiple lymphocyte subset indicators using flow cytometry yielded the following conclusions: Compared with the normal control group, both the commercial thymopeptide group and the recombinant thymopeptide group of the present invention showed a significant increase in double-positive T cells (DPT), indicating that thymopeptide promotes T cell differentiation and maturation and exerts its classic pharmacological effects.

[0085] The treatment group exhibited a unique T cell subset remodeling effect. Despite CD4... + and CD8 + The absolute number of T cells decreased, but thymosin treatment led to a decrease in CD8. + The relative proportion of T cells increased, CD4 + A decrease in the proportion of T cells leads to a decrease in the CD4 / CD8 ratio. This phenomenon is consistent with the known pharmacological effects of natural thymosin, reflecting the differential regulatory capacity of thymosin on different T cell subsets.

[0086] In addition to T cells, recombinant thymosin also significantly regulates the activation and migration of B cells, NK cells, and NKT cells.

[0087] In summary, the recombinant thymosin α1 derived from lactic acid bacteria prepared in this study can significantly induce the proliferation of immature double-positive T cells in vitro; promote the differentiation of double-negative T cells into mature single-positive T cells; and effectively remodel CD4+. + / CD8 + The balance of T cell subsets; comprehensive regulation of the differentiation, distribution, and overall immune pattern of peripheral blood lymphocyte subsets. These results fully demonstrate at the lymphocyte subset level that this recombinant thymopeptide has the ability to mediate T lymphocyte maturation and differentiation and immune lineage remodeling, comparable to commercial products.

Claims

1. A gene for efficiently expressing thymosin α1, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid comprises the gene described in claim 1.

3. A recombinant lactic acid bacteria, characterized in that, The recombinant lactic acid bacteria comprises the recombinant expression plasmid as described in claim 2.

4. The method for inducing efficient expression of thymosin α1 by recombinant lactic acid bacteria as described in claim 3, characterized in that, The induction method was performed at a temperature of 26°C, with a Nisin concentration of 1.5 ng / mL and an induction time of 10 h.

5. A method for preparing thymosin α1, characterized in that, The preparation method includes the induction method described in claim 4.

6. A thymosin α1, characterized in that, The thymosin α1 is prepared by the method of claim 5.

7. The use of thymosin α1 as described in claim 6 in the preparation of immunomodulatory drugs or health products.

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