Genomes, expression cassettes, expression vectors, recombinant strains and uses thereof

Abstract

This invention relates to the field of bioengineering, and more particularly to genomes, expression cassettes, expression vectors, recombinant strains, and their applications. The invention provides a genome including the NGF gene, the malF gene, and the lacZ gene. This invention provides a reliable and efficient system and method for expressing and screening disulfide bond-rich proteins, which not only has broad application prospects in the expression of disulfide bond-rich proteins, but also provides new ideas and methods for research and applications in related fields.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to genomes, expression cassettes, expression vectors, recombinant strains and their applications. Background Technology

[0002] Escherichia coli is a commonly used host for recombinant protein expression, but it presents challenges when expressing proteins containing disulfide bonds. Disulfide bonds are essential for the proper folding and stability of proteins; however, the cytoplasm of E. coli lacks the oxidative environment and cofactors required for the formation of stable disulfide bonds. Therefore, previous expression of disulfide-bonded proteins in E. coli has typically resulted in incomplete folding and aggregation, thereby reducing the activity and solubility of the target protein.

[0003] To address this challenge, scientists have conducted long-term research in an attempt to find solutions suitable for expressing disulfide-bonded proteins in E. coli. This process involves a deep understanding of E. coli cell physiology and protein folding mechanisms, as well as continuous innovative applications of protein engineering and genome editing technologies.

[0004] To overcome the challenge of expressing disulfide bond proteins in *E. coli*, scientists have employed various engineering strategies. One important strategy is to mutate intracellular reduction pathways to promote an oxidative environment that favors disulfide bond formation. This strategy involves mutating key enzymes such as thioredoxin reductase (trxB) and glutathione reductase (gor), reducing the intracellular reduction potential and thus promoting disulfide bond formation.

[0005] In addition, there are several methods for engineering *E. coli*, including altering protein folding pathways, regulating redox balance, and adding cofactors. For example, altering protein folding pathways involves using molecular chaperones such as small thiols to promote isomeric folding and avoid incorrect disulfide bond formation. Regulating intracellular redox balance involves overexpressing genes such as thioreductase to enhance intracellular disulfide bond formation. Introducing cofactors, such as thiotransferases and disulfide isomerases, accelerates the refolding and isomerization of protein disulfide bonds. These methods provide diverse options for *E. coli* to express disulfide bond-containing proteins. However, these methods are mostly based on rational design, resulting in low screening throughput and limited effectiveness. More efficient screening methods are needed to help us obtain superior *E. coli* chassis cells expressing proteins with high disulfide bond formation requirements.

[0006] As part of an engineering strategy, scientists have constructed a series of fusion proteins, such as MalF-LacZ102, to study and optimize the disulfide bond formation mechanism in *E. coli*. The MalF-LacZ102 protein is a fusion product of MalF protein and β-galactosidase (β-Gal), and its effect on disulfide bond formation is studied by altering the protein's subcellular localization. By fusing β-galactosidase with a specific domain of MalF, MalF-LacZ102 can localize β-galactosidase to the *E. coli* cell membrane. This precise subcellular localization provides an important tool for studying the structure and function of cell membrane proteins. The β-galactosidase portion of the MalF-LacZ102 protein exhibits particular sensitivity to disulfide bond formation. When this fusion protein is expressed on the outer membrane of *E. coli* cells, the disulfide bonds of β-galactosidase can form stably due to the favorable oxidative environment of the outer membrane. However, when expressed in cells lacking an active disulfide bond formation system, β-galactosidase cannot form disulfide bonds, leading to its inactivation. Due to its sensitivity to disulfide bond formation, MalF-LacZ102 has become an important tool for studying the disulfide bond formation mechanism in Escherichia coli. By analyzing its expression and activity in different environments, we can delve into the key factors and regulatory mechanisms of the disulfide bond formation pathway in E. coli.

[0007] In recent years, researchers have also used MalF-LacZ102 to construct a screening system for evaluating the activity of disulfide bond-forming enzymes and screening for small molecules that inhibit this enzyme activity. This screening system not only provides an important tool for understanding and regulating the disulfide bond formation mechanism in E. coli, but also offers new ideas and approaches for developing novel drug targets and antibacterial agents.

[0008] In conclusion, engineering E. coli to express disulfide-bonded proteins is a complex and challenging task, involving interdisciplinary research in cell physiology, protein engineering, and biochemistry. However, through continuous efforts and innovation, scientists have made a series of important advances, laying a solid foundation for future research and applications. Summary of the Invention

[0009] In view of this, the present invention provides a genome, expression cassette, expression vector, recombinant strain, and their applications. The present invention provides a reliable and efficient system and method for expressing and screening disulfide bond-rich proteins, which not only has broad application prospects in the expression of disulfide bond-rich proteins, but also provides new ideas and methods for research and applications in related fields.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a genome, including: NGF gene, malF gene and lacZ gene.

[0012] In some embodiments of the present invention, the lacZ gene in the above-described genome is a truncated gene.

[0013] In some embodiments of the present invention, the above-mentioned genome further includes one or more of the following genes: dsbA, dsbB, dsbC, dsbG, trxA, gor, ahpC, surA, fkpA, and hdeB.

[0014] In some embodiments of the present invention, the genome described above further includes one or more of the following genes: dsbA, dsbB, and dsbC.

[0015] In some embodiments of the present invention, the NGF gene in the above-described genome has:

[0016] (1) An amino acid sequence as shown in SEQ ID NO:1; or

[0017] (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or

[0018] (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

[0019] In some embodiments of the present invention, the sequence of SEQ ID NO:1 is: MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQVHWTKLQHSLDTALRRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPREAADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA.

[0020] In some embodiments of the present invention, the sequence encoding the NGF gene in the above-described genome is shown in SEQ ID NO:5.

[0021] The present invention also provides an expression cassette comprising the genome described above.

[0022] In some embodiments of the present invention, the expression cassette further includes one or more of a promoter, a lactose-inducing element, and an IPTG-inducing element.

[0023] In some embodiments of the present invention, the promoter of the above-described expression box includes any one of the following: T7 promoter, tac promoter, or trc promoter.

[0024] In some embodiments of the present invention, the promoter in the above-mentioned expression box is the T7 promoter.

[0025] In some embodiments of the present invention, the lactose operon in the above expression cassette is lacO.

[0026] The present invention also provides an expression vector comprising: the genome described above and / or the expression cassette described above.

[0027] In some embodiments of the present invention, the above-mentioned expression vector includes: expression vector 1 and expression vector 2;

[0028] The expression vector 1 has:

[0029] (4) A nucleotide sequence as shown in SEQ ID NO:2; or

[0030] (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and which has the same or similar function as the nucleotide sequence shown in (4); or

[0031] (6) A nucleotide sequence that has at least 80% identity with the nucleotide sequence shown in (4) or (5); and / or

[0032] The expression vector 2 has:

[0033] (7) A nucleotide sequence as shown in SEQ ID NO:3; or

[0034] (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and which has the same or similar function as the nucleotide sequence shown in (7); or

[0035] (9) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (7) or (8).

[0036]

[0037]

[0038] In some embodiments of the present invention, the expression vector 1 in the above expression vector includes: the malF gene, the lacZ gene, a linker fragment and a backbone plasmid pAC;

[0039] The sequence of the connecting fragment is shown in SEQ ID NO:4: TCGGATCTGATCGAAGGTCGTGGGATCCCC.

[0040] In some embodiments of the present invention, the expression vector 2 described above includes: an NGF gene and a backbone plasmid; the backbone plasmid includes: pET28a or pRSFDute; preferably pET28a.

[0041] In some embodiments of the present invention, the expression vector further includes expression vector 3; the expression vector 3 includes one or more of the following genes: dsbA, dsbB, dsbC, dsbG, trxA, gor, ahpC, surA, fkpA, and hdeB.

[0042] The present invention also provides recombinant strains, which transform and / or transfect the above expression vectors into chassis strains.

[0043] In some embodiments of the present invention, the chassis strain in the above-mentioned recombinant strain includes: Escherichia coli with the lacZ gene knocked out.

[0044] In some embodiments of the present invention, the chassis strain in the above-mentioned recombinant strain includes: BL21(DE3)ΔlacZ or E. coli SHuffleΔlacZ.

[0045] The present invention also provides the use of the above-described genome, expression cassette, expression vector and / or recombinant strain in any of the following:

[0046] (I) Detect the disulfide bond formation ability of the strain; and / or

[0047] (II) Screening for strains containing disulfide bond proteins.

[0048] In some embodiments of the present invention, in the above applications, the screening and / or detection results are obtained based on the detection of the β-lactamase activity of the strain.

[0049] This invention provides an effective screening method for identifying and selecting *E. coli* host strains with strong disulfide bond synthesis capabilities. Through a series of genomic manipulations and activity assays, a strain library with different disulfide bond synthesis capabilities was successfully constructed. After screening and evaluation, these strains ensure high solubility and activity of the target protein / enzyme during expression, thereby improving the efficiency and quality of target protein / enzyme production. This method provides important technical support for the biopharmaceutical field, and is expected to promote research and application development in related fields, laying a solid foundation for new drug development and innovative treatment methods. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0051] Figure 1 Comparison of N1 and N2 β-lactamase activities;

[0052] Figure 2 Comparison of the activity / OD600 of N1, N2 and cofactor-expressing strains;

[0053] Figure 3 Comparison of soluble levels of N1-N5 neurotrophic factor expression; where: W represents whole bacteria, S represents supernatant, and P represents precipitate;

[0054] Figure 4 Comparison of NGF activity. Detailed Implementation

[0055] This invention discloses genomes, expression cassettes, expression vectors, recombinant strains, and their applications.

[0056] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0057] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0058] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0059] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0060] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0061] All genome manipulations, knock-in, and knock-outs disclosed herein were performed using a site-specific recombination protocol derived from Kirill A. Datsenko and Barry L. Wanner. This protocol is a standard molecular cloning technique well-known to those skilled in the art.

[0062] This invention discloses a highly efficient screening system for heterologous expression of Escherichia coli rich in disulfide bond proteins based on β-galactosidase, as well as the construction method and application of the system.

[0063] First, this invention relates to the construction of a disulfide bond-rich protein expression system. Taking human nerve growth factor (NGF) as an example, the NGF gene (accession number AAA59931.1) was codon-optimized and cloned into an expression vector such as pET28a. This vector contains a T7 promoter and an IPTG inducible element to ensure high-level NGF expression under induction conditions. The optimized NGF gene sequence is shown in SEQ ID NO:1.

[0064] Secondly, this invention discloses a method for constructing a highly efficient screening system for heterologous expression of disulfide bond-rich Escherichia coli based on β-galactosidase. This system utilizes truncated β-galactosidases (β-Galdbs) expressed in the outer cell membrane and leverages their sensitivity to disulfide bond formation to successfully screen recombinant strains with different disulfide bond formation capabilities.

[0065] Specifically, the DNA sequence of the malF gene (accession number AKK14279.2) was ligated with the DNA sequence of the truncated N-terminal 102 amino acids of the lacZ gene (accession number STK60809.1) and placed in the T7 promoter expression cassette regulated by lacO. The ligated DNA sequence was then inserted into plasmid pAC to obtain plasmid pAC-Galdbs, the specific sequence of which is shown in SEQ ID NO:2.

[0066] Then, *Escherichia coli* BL21(DE3)ΔlacZ and *E. coli* SHuffleΔlacZ strains were transformed and named E1 and E2, respectively. pAC-Gal dbs The plasmids were introduced into strains E1 and E2, and the disulfide bond formation capacity of the two host strains was assessed by β-galactosidase activity assay.

[0067] In this invention, the optimized NGF gene is introduced into E1 and E2 strains, and the corresponding N1 and N2 strains are obtained by transformation.

[0068] By introducing different combinations of cofactors into strain N1, the disulfide bond formation capacity of strains containing different cofactors was detected using high-throughput assays. Three strains (N3, N4, and N5) with the lowest β-lactamase activity / OD600 ratio were selected.

[0069] Comparison of NGF expression levels in these strains showed that the expression levels of N3-N5 were significantly higher than those of N1 and N2, demonstrating the important application value of the screening system in finding and optimizing hosts rich in disulfide bond proteins.

[0070] In Examples 1 to 4 of this invention, all raw materials and reagents used can be purchased from the market.

[0071] The present invention will be further illustrated below with reference to the embodiments:

[0072] Example 1: Construction of a β-galactosidase activity screening system

[0073] 1. Genome manipulation: Gene exchange at specific sites using specific inverted repeat sequences (e.g., the Cre-LoxP system). By briefly exposing to Cre recombinase, selectable marker genes are eliminated while the target gene is integrated into the genome.

[0074] For knockout, the deletion cassette consists of selectable markers flanked by loxP sites. Gene knockout is achieved by transiently exposing the gene to Cre recombinase, thereby eliminating the selectable marker genes.

[0075] Gene knockout, such as lacZ, can be performed as follows: A PCR amplicon is generated containing an antibiotic resistance gene flanked by two FRT sites and homology extensions (H1 and H2), the homology extensions being homologous to the ends of the gene to be knocked out. After transforming cells with this PCR product, the gene to be knocked out is replaced with the antibiotic resistance gene through Red-mediated recombination in these flanking homology regions. After selection, the resistance gene can be eliminated using a helper plasmid expressing FLP recombinase, which acts on the directly repeated FRT (FLP recognition target) sites flanked by the resistance gene. The Red and FLP helper plasmids can be easily handled by growing at 37°C, as they are temperature-sensitive replicons.

[0076] 2. Plasmid construction: The DNA sequence of the malF gene (NCBI accession number AKK14279.2) was ligated with the DNA sequence (TCGGATCTGATCGAAGGTCGTGGGATCCCC as shown in SEQ ID NO:4) and the DNA sequence of the truncated N-terminal 102 amino acids of the lacZ gene (NCBI accession number STK60809.1) and placed in the T7 promoter expression cassette regulated by lacO. The plasmid was synthesized by DNA synthesis company (Genewiz Biotechnology Co., Ltd.) and inserted into the commonly used plasmid pAC. The specific sequence is shown in SEQ ID NO:2.

[0077] Comparative strains and nomenclature: In the examples, the *E. coli* BL21(DE3)ΔlacZ strain was obtained by knocking out the lacZ gene using CRISPR technology and used as a control strain. Simultaneously, the *E. coli* SHuffleΔlacZ strain was introduced as another control strain. The *E. coli* SHuffle strain was purchased from New England Biolab (NEB), catalog number C3026J. Both strains were transformed into pAC-Gal... dbs They were named E1 and E2 respectively.

[0078] Human nerve growth factor (NGF) (accession number AAA59931.1) (as shown in SEQ ID NO:1) was cloned into expression vectors such as pET28a and pRSFDute, with pET28a being preferred. This vector contains promoter sequences such as the T7 promoter, tac promoter, and trc promoter, with the T7 promoter being preferred, and inducible elements such as the IPTG inducible element and the lactose inducible element, to ensure high-level expression of the target protein under induction conditions.

[0079] First, the codon-optimized NGF gene was ligated to the pET28a vector by double enzyme digestion. The two were then precisely ligated using restriction enzymes, as shown in SEQ ID NO:3. The ligated recombinant plasmid was then transformed in E. coli using either heat shock freezing or electroporation to ensure stable transformation.

[0080] The transformed bacterial colonies were subjected to PCR verification and sequencing to confirm the correct insertion of the NGF gene and the integrity of the plasmid. Once verification was successful, the correct plasmid was used for large-scale culture, yielding the target plasmid pNGF.

[0081] The NGF expression plasmid was transformed into strains E1 and E2, respectively, to obtain NGF expression strains N1 and N2.

[0082] 3. Culture conditions and induction: The constructed strain was cultured in LB medium containing appropriate antibiotics until the mid-log phase. Subsequently, IPTG was added to the medium at 37°C to a final concentration of 0.1 mM, and the culture was continued at 30°C for 6 hours to induce the expression of the target gene.

[0083] 4. Activity detection:

[0084] (1) Prepare M63 medium containing essential amino acids but no cysteine, and add appropriate antibiotics.

[0085] (2) Transfer the cultured strain to a 96-well plate in a volume of 200 μL and dilute it twice.

[0086] (3) Perform a two-fold dilution of IPTG on the well plate to conduct activity tests on multiple strains.

[0087] (4) Use Synergy H1 for 10 hours of rapid orbital oscillation culture at 30℃.

[0088] (5) After culturing, the growth of the strain was determined by measuring the absorbance at 600 nm.

[0089] (6) Transfer the strains on the growth plate to the enzyme activity detection plate without cell lysis.

[0090] (7) The reaction was initiated by adding a buffer containing ONPG, and the absorbance at 420 nm was measured to track the kinetics of ONPG hydrolysis.

[0091] 5. Data Analysis: Data analysis was performed using GraphPad Prism software to calculate reaction rates and use them to calculate Miller units to evaluate β-galactosidase activity. The β-lactamase activities of different strains were compared to assess the impact of different host disulfide bond formation capabilities on β-galactosidase activity.

[0092] 6. Experimental results: such as Figure 1 As shown, the β-lactamase activity of N1 strain, an NGF-expressing strain of the E. coli BL21(DE3)ΔlacZ host, was 427.3, while the β-lactamase activity of N2 strain, an NGF-expressing strain of the E2 strain of the SHuffleΔlacZ host with stronger disulfide bond formation ability, was 275.1. The difference between the two is statistically significant, demonstrating that this system can use β-lactamase activity to indicate the host's disulfide bond formation ability.

[0093] Example 2: Strains obtained by screening the auxiliary factor library

[0094] In this embodiment, we optimize the characteristics of the target strain by screening genes in the helper factor library that may enhance the disulfide bond formation ability of Escherichia coli.

[0095] The steps are as follows:

[0096] Cofactors associated with disulfide bond formation were identified. These factors included known enzymes and cofactors that promote protein folding and improve redox balance. The following cofactors were ligated into the pGEX-5x plasmid via BamHI and XhoI sites and transformed into strain N1, resulting in strains N3, N4, N5, N6, N7, N8, N9, N10, N11, and N12.

[0097] Monoclonal clones N1-N12 were transferred to cysteine-free M63 medium and cultured until OD600 = 0.4. IPTG was then added to a final concentration of 0.1 mM, and the clones were cultured at 30°C for another 4 h. The β-lactamase activity and OD600 of each monoclonal clone were determined using the aforementioned method. 600 The ratio was calculated, and the three single clones with the lowest ratios were selected.

[0098] Subsequently, the initially screened strains were repeatedly validated. The performance stability of these strains was confirmed through multiple independent experiments, and the best-performing strains were further screened. Ultimately, strains N3, N4, and N5 were selected as the most stable strains with the best lactamase activity. These strains will serve as high-quality expression hosts for subsequent studies. The enzyme activity / OD ratios of N3, N4, and N5 were 109.9, 119.8, and 126.1, respectively, lower than those of N1 and N2 (427.3 and 275.1).

[0099] Table 1 Enzyme activity / OD600 ratio

[0100]

[0101] Example 3: Effects of different hosts on human nerve growth factor expression

[0102] In the expression experiments, strains N1 to N5 were cultured in M63 medium containing essential amino acids but cysteine, with appropriate antibiotics added (kanamycin 50 mg / L, chloramphenicol 25 mg / L). The strains were cultured at 3°C ​​for 12-16 hours after transformation.

[0103] After reaching the logarithmic growth phase, strains N1 to N5 were induced with IPTG to enable the T7 promoter to initiate the transcription and translation of the NGF gene. The final IPTG concentration was 0.1 mM. Parameters such as induction time, temperature, and light intensity will be optimized according to experimental needs to achieve the best NGF expression level.

[0104] Following IPTG induction, the bacterial strain is cultured for an additional 6-8 hours. After expression is complete, the cells are collected by centrifugation, and the cell supernatant is obtained using an appropriate cell lysis method (such as sonication or chemical lysis) to extract the expressed NGF protein.

[0105] The extracted NGF protein can be analyzed by SDS-PAGE gel electrophoresis to assess the expression level and purity of NGF. Further detection and identification of the protein can be performed using methods such as Western blot.

[0106] Finally, comparative analysis of NGF protein expression in strains N1 to N5 showed that the expression levels of N3-N5 were significantly higher than those of N1 and N2. Figure 3 (This is used for subsequent NGF production and application research.)

[0107] Example 4: Assay of NGF activity in different hosts

[0108] This embodiment aims to perform an activity test on partially purified NGF to evaluate its function in promoting neuronal growth.

[0109] 1. Strains culture and protein expression: Strains N1 to N5 carrying the pET-NGF plasmid were cultured separately in LB medium (400 mL × 2 per medium), and the culture and induction methods were the same as before. Cells were collected and washed with 0.85% NaCl, and 4 g of wet cells were suspended in 40 mL of buffer containing a 0.1% (v / v) protease inhibitor mixture (Sigma).

[0110] 2. Preparation of vacuolar components: Cellular vacuolar components were prepared and concentrated using a Centiprep 10 concentrator (Amicon). The buffer solution was adjusted to contain 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA, and then resonated overnight with Affi-prep Polymixin Matrix (Bio-Rad) at 4°C using a rotary shaker. After centrifugation at 10,000 × g for 10 min, the supernatant was subjected to ion exchange chromatography on a DEAE Toyopearl column (Tosoh, Tokyo, Japan). The eluent was concentrated to ~2 mL and dialyzed against 20 mM Tris-HCl (pH 8.0) and 1 mM EDTA.

[0111] 3. Activity Assay of Partially Purified NGF: Partially purified NGF samples were added to rat PC12 cells grown in 24-well collagen-coated culture dishes for activity assay. The culture medium was RPMI 1640 (Life Technologies, Inc.) supplemented with 5% fetal bovine serum and 10% horse serum. Mouse NGF (Biomedical Technologies, Inc.) was used as the standard for comparison. After incubating the culture dishes at 37°C and 5% humidified CO2 for 7 days, the neuronal processes were observed under a microscope.

[0112] 4. Results Analysis: The activity level of NGF samples was evaluated by comparing the number and length of neurites induced in PC12 cells by NGF samples with that of mouse NGF standard. The cell response to NGF was calculated based on the average percentage of neurite cells in three microscopic fields to determine the activity of NGF samples. The results showed that the NGF activity expressed by strains N3 and N5 was comparable to that of mouse neurotrophic factor (100 ng / mL) (as shown in Table 2). Figure 4 (As shown).

[0113] Table 2. Synaptic percentage (%)

[0114]

[0115] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A genome, characterized by, include: NGF gene, malF gene and lacZ gene.

2. The genome as described in claim 1, characterized in that, Also includes: One or more of the following genes: dsbA, dsbB, dsbC, dsbG, trxA, gor, ahpC, surA, fkpA, and hdeB.

3. The genome as described in claim 1 or 2, characterized in that, The NGF gene has the following characteristics: (1) An amino acid sequence as shown in SEQ ID NO:1; or (2) An amino acid sequence obtained by substituting, deleting, or adding one or more amino groups to the amino acid sequence shown in (1), and which has the same or similar function as the amino acid sequence shown in (1); or (3) An amino acid sequence that is at least 80% identical to the amino acid sequence shown in (1) or (2).

4. An expression box, characterized in that, include: The genome as described in any one of claims 1 to 3.

5. The expression box as described in claim 4, characterized in that, Also includes: One or more of the following: promoter, lactose inducible element, and IPTG inducible element.

6. An expression carrier, characterized in that, include: The genome as described in any one of claims 1 to 3 and / or the expression cassette as described in claim 4 or 5.

7. The expression vector as described in claim 6, characterized in that, include: Expression vector 1 and expression vector 2; The expression vector 1 has: (4) A nucleotide sequence as shown in SEQ ID NO:2; or (5) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (4), and which has the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence that has at least 80% identity with the nucleotide sequence shown in (4) or (5); and / or The expression vector 2 has: (7) A nucleotide sequence as shown in SEQ ID NO:3; or (8) A nucleotide sequence obtained by substituting, deleting, or adding one or more bases to the nucleotide sequence shown in (7), and which has the same or similar function as the nucleotide sequence shown in (7); or (9) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (7) or (8).

8. A recombinant strain, characterized in that, The expression vector as described in claim 6 or 7 is transformed and / or transfected into the chassis strain.

9. The recombinant strain according to claim 8, characterized in that, The chassis strains include: Escherichia coli with the lacZ gene knocked out.

10. The use of the genome as described in any one of claims 1 to 3, the expression cassette as described in claim 4 or 5, the expression vector as described in claim 6 or 7, and / or the recombinant strain as described in claim 8 or 9 in any of the following: ( ), and detect the disulfide bond formation ability of the strain; and / or ( ), and screen for strains containing disulfide bond proteins.

11. The application as described in claim 10, characterized in that, The screening and / or the detection are based on the detection of the β-lactamase activity of the strain.

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