Protein for enhancing foreign protein expression and application thereof

Abstract

The invention discloses a protein for enhancing foreign protein expression and application thereof. The target protein-OMP43 recombinant protein can be obtained by fusing and connecting the protein OMP43 to the C end of the target protein, so that the expression level of the target protein in a heterologous expression system is improved. The recombinant protein En6-OMP43 is constructed and obtained by taking bacteriophage lyase En6 as an example. Experimental results show that after the OMP43 is fused, the antibacterial activity of the recombinant protein lyase on acinetobacter baumannii is maintained. The protein OMP43 serving as an expression enhancing element is high in universality, simple and convenient to construct and suitable for being modularly applied to foreign protein expression and preparation.

Description

Technical Field

[0001] This invention belongs to the field of proteins, specifically relating to a protein that enhances the expression of exogenous proteins and its applications. Background Technology

[0002] In recent years, there has been a significant increase in severe hospital-acquired infections caused by Gram-negative opportunistic pathogens. Among these, *Acinetobacter baumannii* has attracted widespread attention due to its strong long-term survival ability in medical environments such as ICUs, its tendency to form biofilms, and its ability to cause refractory infections in various clinical scenarios, including ventilator-associated pneumonia, bacteremia, and wound infections. More seriously, *Acinetobacter baumannii* readily develops resistance to multiple classes of antimicrobial drugs, leading to a continuous decline in the effectiveness of traditional antibiotic treatments. Therefore, there is an urgent need to develop new anti-infection strategies and alternative / complementary treatments.

[0003] In the research of novel antimicrobial strategies, phage therapy and phage-derived antimicrobial products are considered important directions in combating drug-resistant bacteria. Phages possess potential advantages such as high host specificity and low cytotoxicity to human cells, but in practical applications, they may also face challenges such as rapid bacterial development of phage resistance, consistency of formulation quality, and complex regulatory pathways. To overcome these limitations, increasing research is turning to phage-derived antimicrobial effector molecules, especially phage lyases that can directly act on the bacterial cell wall structure. Lyases generally disrupt the cell wall by hydrolyzing specific chemical bonds in peptidoglycan, leading to bacterial lysis and death; furthermore, lyases can be engineered to optimize their properties, possessing the potential to serve as novel antimicrobial agents, disinfectants, or components of antimicrobial materials.

[0004] Despite the attractiveness of lyases in the fight against drug-resistant bacteria, their transformation from laboratory research to application typically requires addressing three key challenges: First, the outer membrane barrier of Gram-negative bacteria hinders lyase secretion. Traditional signal peptides (such as OmpA and PelB) have a transmembrane efficiency of <30%, preventing intracellularly retained lyases from reaching extracellular pathogens. Furthermore, purification processes require cell disruption, increasing costs. Protein modification or formulation strategies are needed to improve efficacy. Second, as protein drug candidates, the research and industrialization of lyases heavily rely on high-quality, reproducible recombinant protein preparation processes. The expression level and solubility of exogenous proteins in heterologous expression systems often constitute substantial bottlenecks. Lyases often possess specific domain combinations, charged characteristics, or locally hydrophobic / aggregating fragments, leading to low expression levels, inclusion body formation, insufficient solubility, and protein degradation in commonly used prokaryotic expression hosts (such as *E. coli*). This increases purification costs and affects activity evaluation and formulation development. Third, reduced activity is a concern. Forced secretion strategies (such as Sec pathway enhancement) often result in protein hydrolysis or structural distortion, reducing bactericidal potency.

[0005] For proteins that are difficult to express, existing technologies have formed a relatively systematic solution path, mainly including: codon optimization, promoter and RBS optimization, reducing induction intensity and temperature, co-expression molecular chaperones, changing host strains, optimizing culture medium and fermentation parameters, and fusion expression tags. Among them, the fusion tag strategy is widely used due to its convenient construction and wide applicability: by fusing the target protein with a specific tag protein or expression-promoting fragment, translation initiation can be improved to a certain extent, folding can be promoted, solubility can be increased, and purification can be simplified. For example, small ubiquitin-like modifier (SUMO) tags are widely used because of their folding stability and ability to improve the solubility of certain target proteins. CN113025598A discloses a method for preparing recombinant heparinase III using a SUMO fusion expression system. However, traditional fusion tag strategies still have significant limitations in practical applications. First, commonly used solubilizing / expression-promoting tags (such as MBP, GST, NusA, etc.) often have large molecular weights, which can significantly increase the overall size of the fusion protein and may affect the conformation and function of the target protein. Moreover, when preparing tagless target proteins, it is usually necessary to introduce additional enzyme cleavage sites and perform cleavage and secondary purification, which is complex and costly. Secondly, different tags exhibit significantly different enhancement effects on different target proteins, requiring repeated construction, screening, and condition optimization. Thirdly, for enzyme proteins with fine structure-function relationships, factors such as the location of the fusion tag (N-terminus or C-terminus), spatial interference between the tag and the target protein, and the accessibility of post-expression cleavage all affect the final activity recovery.

[0006] Therefore, developing an expression-enhancing peptide element with smaller molecular weight, simpler construction, less impact on the activity of the target protein, and stable ability to improve expression levels, and using it for the fusion expression and preparation of antimicrobial proteins such as phage lysins, is of great significance for reducing R&D and production costs, improving protein preparation efficiency, and promoting the development of products related to the prevention and control of drug-resistant bacteria. Summary of the Invention

[0007] In order to overcome the above-mentioned shortcomings of the existing technology, the purpose of this invention is to provide a protein OMP43 that can universally promote the expression of the target protein and has little impact on the biological activity of the target protein, and its application, so as to achieve the efficient preparation of exogenous proteins (especially antimicrobial proteins such as phage lysins). It can simultaneously solve the three major technical problems of expression level, secretion rate and activity maintenance, and overcome the limitations of low efficiency of traditional signal peptides and the increased metabolic burden of molecular chaperones.

[0008] The objective of this invention is achieved by at least one of the following technical solutions.

[0009] A first aspect of the present invention provides a protein OMP43 comprising any one of the amino acid sequences shown in (1)-(2): (1) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:4 and has the same function; (2) An amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO:4.

[0010] In some embodiments, protein OMP43 has an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO:4, for example, at least 95%, preferably at least 96%, even more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, and more preferably at least 99.9% homologous to the amino acid sequence shown in SEQ ID NO:4, and the protein having the above-mentioned homologous amino acid sequence has the same function as the protein shown in SEQ ID NO:4.

[0011] In some embodiments, protein OMP43 has an amino acid sequence obtained by modifying, substituting, deleting, or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO:4, and the protein having the above amino acid sequence has the same function as the protein shown in SEQ ID NO:4 (promoting the expression of exogenous proteins).

[0012] In a preferred embodiment, the protein OMP43 has the amino acid sequence shown in SEQ ID NO:4.

[0013] A second aspect of the present invention provides a nucleic acid molecule that encodes the protein OMP43 described in any one of the first aspects.

[0014] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0015] A third aspect of the present invention provides a recombinant protein comprising a target protein and the protein OMP43 described in any one of the first aspects, wherein the protein OMP43 is fused to the target protein to improve the expression level of the target protein in a heterologous expression system.

[0016] In some embodiments, the protein OMP43 is fused to the C-terminus of the target protein.

[0017] In some embodiments, a flexible linker is present between the target protein and protein OMP43.

[0018] In some preferred embodiments, the amino acid sequence of the flexible linker is AGAGAG.

[0019] In some embodiments, the target protein includes phage lysin En6; the amino acid sequence of phage lysin En6 is shown in SEQ ID NO:2, and the encoding nucleotide sequence of phage lysin En6 is shown in SEQ ID NO:1.

[0020] In some preferred embodiments, the recombinant protein includes recombinant protein En6-OMP43; the amino acid sequence of recombinant protein En6-OMP43 is shown in SEQ ID NO:6, and the encoding nucleotide sequence of recombinant protein En6-OMP43 is shown in SEQ ID NO:5.

[0021] A fourth aspect of the present invention provides a recombinant nucleic acid molecule that encodes the recombinant protein described in any of the third aspects.

[0022] The nucleotide sequence of the recombinant nucleic acid molecule is shown in SEQ ID NO:5.

[0023] A fifth aspect of the present invention provides a recombinant expression vector comprising the recombinant nucleic acid molecule described in any of the fourth aspects.

[0024] In some embodiments, the recombinant expression vector described above may further include an expression regulatory sequence operatively linked to the sequence of the recombinant nucleic acid molecule to facilitate protein expression.

[0025] A sixth aspect of the present invention provides a host cell comprising the recombinant expression vector described in any of the fifth aspects.

[0026] In some embodiments, the host cell includes prokaryotic and eukaryotic cells used for cloning or expressing DNA. Commonly used prokaryotic host cells include, but are not limited to, *Escherichia coli*, *Bacillus subtilis*, etc., for example, *E. coli* cells, such as *E. coli* HMS174 (DE3) or BL21 (DE3). Commonly used eukaryotic host cells include, but are not limited to, yeast cells, insect cells, mammalian cells, etc.

[0027] The seventh aspect of the present invention provides the use of the protein OMP43 described in any of the first aspects in improving the expression level of a target protein.

[0028] In some embodiments, the application is to improve the expression level of the target protein in a heterologous expression system by linking the protein OMP43 to the target protein in a fusion manner (preferably at the C-terminus and linked by a flexible linker).

[0029] The eighth aspect of the present invention provides the use of the recombinant protein according to any one of the third aspects in the preparation of a medicament for the prevention and / or prevention of Acinetobacter baumannii infection. Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The protein OMP43 of the present invention can be used as an expression enhancement element for heterologous expression of exogenous proteins, which can promote the expression level of the target protein, increase the amount of the target protein obtained, thereby reducing the cost of expression screening and preparation, and improving production efficiency.

[0030] (2) The protein OMP43 of this invention has little impact on the biological function of the target protein, can maintain its activity, and exhibits superior antibacterial ability in some recombinant proteins. After OMP43 is fused to the C-terminus of the target protein through a flexible linker (e.g., AGAGAG), the functional activity of the target protein can remain stable. Taking phage lysin as an example, turbidimetric results show that the bactericidal / bacteriostatic effects of En6-OMP43 and En6, and En8-OMP43 and En8 on Acinetobacter baumannii are basically the same, indicating that OMP43 fusion does not have an adverse effect on the activity of the lysin; the in vitro MIC determination results show that En6, En6-OMP43, En8 and En8-OMP43 all have antibacterial effects on Acinetobacter baumannii, indicating that the OMP43 fusion construct can achieve effective antibacterial effect while maintaining the antibacterial function of the lysin. Taking En6 as an example, the MIC decreases after fusion with OMP43, and the recombinant protein En6-OMP43 has better antibacterial performance.

[0031] (3) The protein OMP43 of the present invention is highly versatile, easy to construct, and suitable for modular application. OMP43 is a short peptide fragment, which is convenient to use in combination with different target proteins through gene fusion. If a flexible linker is set between the target protein and OMP43, steric hindrance and conformational interference can be reduced. This strategy has good scalability and versatility and is suitable for the expression enhancement and preparation of a variety of exogenous proteins. Attached Figure Description

[0032] Figure 1 The diagram shows the construction patterns of the recombinant plasmids pET28a-En6, pET28a-En6-OMP43, pET28a-En8, and pET28a-En8-OMP43 from Example 1.

[0033] Figure 2 The images show the gel electrophoresis band patterns of the recombinant proteins in each group in Example 2.

[0034] Figure 3 The following are line graphs showing the in vitro bactericidal activity results of the recombinant proteins in Example 4. Specifically, A is a line graph comparing the in vitro bactericidal activity results of protein En6 and recombinant protein En6-OMP43; B is a line graph comparing the in vitro bactericidal activity results of protein En8 and recombinant protein En8-OMP43. Detailed Implementation

[0035] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0036] In this invention, the term "homology" refers to the overall correlation between polymer molecules, such as between nucleic acid molecules (e.g., DNA and / or RNA molecules) and / or between polypeptide molecules. Generally, the term "homology" implies an evolutionary relationship between two molecules. Therefore, two homologous molecules will share a common evolutionary ancestor. In the context of this invention, the term homology includes both identity and similarity.

[0037] "Homology" can be determined using methods known in the art, such as sequence comparison algorithms, where two or more sequences have a specified percentage of identical nucleotides in a specified region when compared and aligned at maximum consistency on a comparison window. The percentage of "homology" between two sequences can be determined using BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.) with default parameters.

[0038] A polymer molecule is considered “homological” if at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of its monomers are identical (completely identical monomers) or similar (conservative substitutions). The term “homological” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).

[0039] In this invention, the terms “protein” or “polypeptide” or “peptide” are used interchangeably to refer to any chain of two or more natural or non-natural amino acids, whether or not they have been post-translational modified (e.g., glycosylated or phosphorylated), constituting all or part of a naturally occurring or non-natural polypeptide or peptide.

[0040] As used in this invention, the term "nucleic acid" is intended to include polymeric forms of nucleotides of any length containing deoxyribonucleotides, ribonucleotides, and / or their analogues, including DNA, RNA, and DNA / RNA hybrids, and also including DNA or RNA analogues, such as those containing a modified backbone (e.g., peptide nucleic acid (PNA) or phosphate thioester) or modified bases. Therefore, the nucleic acids of this invention include DNA, cDNA, mRNA, recombinant nucleic acids, etc.

[0041] Example 1: Construction of engineered expression strains En6, En6-OMP43, En8, and En8-OMP43 Based on the codon preference of E. coli, the nucleic acid sequence encoding the phage lysin En6 (SEQ ID NO:1) and the En6-OMP43 nucleotide sequence (SEQ ID NO:5) with the OMP43 peptide fused to the C-terminus of the En6 protein (SEQ ID NO:3) were designed and synthesized. En6 and OMP43 are connected by a flexible linker (amino acid sequence: AGAGAG). The nucleic acid sequence encoding the phage lysin En8 (SEQ ID NO:7) and the En8-OMP43 nucleotide sequence (SEQ ID NO:9) with the OMP43 peptide fused to the C-terminus of the En8 protein were synthesized. En8 and OMP43 are connected by a flexible linker (amino acid sequence: AGAGAG). Using the ClonExpress Ultra One Step Cloning Kit from Nanjing Novizan Biotechnology Co., Ltd., the synthetically produced nucleotide sequences of En6, En6-OMP43, En8, and En8-OMP43 were seamlessly ligated between the Nco I and Hind III restriction sites of the expression vector pET28a(+), respectively, to obtain the recombinant expression plasmids pET28a-En6, pET28a-En6-OMP43, pET28a-En8, and pET28a-En8-OMP43. Figure 1 As shown. Each recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells to obtain engineered strains expressing the recombinant proteins pET28a-En6, pET28a-En6-OMP43, pET28a-En8, and pET28a-En8-OMP43. The transformation methods are as follows: 1) Add 1 μL of the recombinant plasmid to be transformed into an EP tube containing 100 μL of BL21(DE3) competent cells, gently rotate a few times to mix the contents, and incubate on ice for 30 min; 2) After heat shocking the bacteria in a 42 ℃ water bath for 90 seconds, immediately return them to an ice bath to cool them down for 2 minutes. 3) Add 800 μl of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) to each tube and incubate with gentle shaking at 37°C for 45 min to allow the cells to recover; 4) Centrifuge at low speed for 5 min, discard the supernatant, retain about 100 μl of culture medium in the centrifuge tube, and resuspend the bacterial cells; 5) Spread the bacterial suspension evenly on LB agar plates containing kanamycin; incubate upside down at 37 ℃, and observe for colonies after 12-16 h.

[0042] SEQ ID NO:1 (nucleotide sequence of En6) ATGGCTAAACTGGATAAAAACGTGCAGCGCAAAGCGTTTCTGGATATGATTGCGTATAGCGAAGGCACCGATAACGGCCGTCAGCCGACCAAAGATCGCGGCTATGATGTGATTGTGGGCGGCAAACTGTTTAGCGACTATAGCAAACATCCGGGCGTGTATGTGAAACTGAACGCGAAACTGACGAGCAGCGCGGCGGGCCGCTATCAGATTCTGGAAAAATTTGCGAAACACTAT ATGGAACAGCTGGGCCTGCCGGATTTTGGCCCGGCGAGCCAAGATAAAATTGCGCTGCAGCTGATTCGCGAATGCAAAGCGCTGACCGATATTGATGAAGGCCGCATTCATGAAGCGATTCGCAAATGCCGCAGCCGCTGGGCGAGCCTGCCGGGCGGGCTATGGTCAGCATGAACAGAAAATTGAAAAACTGATTGAAGTGTTTAAAAAAGCGGGCGGCATTGTGCTGGATTAA SEQ ID NO:2 (Amino acid sequence of En6) MAKLDKNVQRKAFLDMIAYSEGTDNGRQPTKDRGYDVIVGGKLFSDYSKHPGVYVKLNAKLTSSAAGRYQILEKFAKHYMEQLGLPDFGPASQDKIALQLIRECKALTDIDEGRIHEAIRKCRSRWASLPGAGYGQHEQKIEKLIEVFKKAGGIVLD SEQ ID NO:3 (nucleotide sequence of OMP43) AAATTCCATGAAAAACATCACTCCCACCGTGGTTAC SEQ ID NO:4 (Amino acid sequence of OMP43) KFHEKHHSHRGY SEQ ID NO:5 (Nucleotide sequence of En6-OMP43) ATGGCTAAACTGGATAAAAACGTGCAGCGCAAAGCGTTTCTGGATATGATTGCGTATAGCGAAGGCACCGATAACGGCCGTCAGCCGACCAAAGATCGCGGCTATGATGTGATTGTGGGCGGCAAACTGTTTAGCGACTATAGCAAACATCCGGGCGTGTATGTGAAACTGAACGCGAAACTGACGAGCAGCGCGGCGGGCCGCTATCAGATTCTGGAAAAATTTGCGAAACACTATATGGAACAGCTGGGCCTGCCGGATTTTGGCCCGGCGAGCCAAGATAAAATTGCGCTGCAGCTGATTCGCGAATGCAAAGCGCTGACCGATATTGATGAAGGCCGCATTCATGAAGCGATTCGCAAATGCCGCAGCCGCTGGGCGAGCCTGCCGGGCGCGGGCTATGGTCAGCATGAACAGAAAATTGAAAAACTGATTGAAGTGTTTAAAAAAGCGGGCGGCATTGTGCTGGATGCGGGTGCTGGTGCGGGTAAATTCCATGAAAAACATCACTCCCACCGTGGTTACTAA SEQ ID NO:6 (Amino acid sequence of En6-OMP43) MAKLDKNVQRKAFLDMIAYSEGTDNGRQPTKDRGYDVIVGGKLFSDYSKHPGVYVKLNAKLTSSAAGRYQILEKFAKHYMEQLGLPDFGPASQDKIALQLIRECKALTDIDEGRIHEAIRKCRSRWASLPGAGYGQHEQKIEKLIEVFKKAGGIVLDAGAGAGKFHEKHHSHRGY SEQ ID NO:7 (Nucleotide sequence of En8) ATGAAAACGAGCAACCCGGGCGTGGATCTGATTAAAGGCTTTGAAGGCCTGCGCCTGAAAGCGTATGATGATGGCGTGGGCGTGTGGACCATTGGCTTTGGCACCATTAAATATCCGAACGGCGTGCGCGTGAAAAAAGGCGATACCTGCACCGAAAGCCAAGCGGAAGAATATCTGCGCAACGATCTGGTGGTGTTTGAAAGCGCGATTAACCGCCTGGTGAAAGTGCCGCTGAATCAGAATCAGTTTGATGCGCTGGCGAGCTTTACCTATAACCTGGGCGAAGGCAACCTGAGCATTAGCACCCTGCTGAAAAAACTGAACGCGAAAGACTATAAAGGCGCGGCCGCGGAATTTCCGAAATGGAACAAAGCGGGCGGCCGCGTGCTGGCGGGCCTGGTGAAACGCCGCAAAGCGGAAATGGAACTGTTTCTGAAAAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTGTAA SEQ ID NO:8 (Amino acid sequence of En8) MKTSNPGVDLIKGFEGLRLKAYDDGVGVWTIGFGTIKYPNGVRVKKGDTCTESQAEEYLRNDLVVFESAINRLVKVPLNQNQFDALASFTYNLGEGNLSISTLLKKLNAKDYKGAAAEFPKWNKAGGRVLAGLVKRRKAEMELFLKNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKGAKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKL SEQ ID NO:9 (Nucleotide sequence of En8 - OMP43) ATGAAAACGAGCAACCCGGGCGTGGATCTGATTAAAGGCTTTGAAGGCCTGCGCCTGAAAGCGTATGATGATGGCGTGGGCGTGTGGACCATTGGCTTTGGCACCATTAAATATCCGAACGGCGTGCGCGTGAAAAAAGGCGATACCTGCACCGAAAGCCAAGCGGAAGAATATCTGCGCAACGATCTGGTGGTGTTTGAAAGCGCGATTAACCGCCTGGTGAAAGTGCCGCTGAATCAGAATCAGTTTGATGCGCTGGCGAGCTTTACCTATAACCTGGGCGAAGGCAACCTGAGCATTAGCACCCTGCTGAAAAAACTGAACGCGAAAGACTATAAAGGCGCGGCCGCGGAATTTCCGAAATGGAACAAAGCGGGCGGCCGCGTGCTGGCGGGCCTGGTGAAACGCCGCAAAGCGGAAATGGAACTGTTTCTGAAAAATAGTACCTCTAATAGTAGCACCAATTCAGGCTCTACCGGTAAAGTGAGTCTGCCGAATCGTGTGATTCGCGTGACCAAACCGATTGTTCATGGTAGCGATGTGCTGGCAATTCAGAAAGCACTGTCAAGCCTGTATTTTTATCCGGAAAAAGGTGCCAAAGATAATGGTTCCGATAGCTATTATGGTCCGAAAACCGCCAATGCCGTTAAACGCTTTCAGTCTGTTAATGGCTTAGTTGCAGATGGCGTGTATGGCCCGAAAACCCGCGCAGCCATTCTGAAAAAACTGGCGGGTGCTGGTGCGGGTAAATTCCATGAAAAACATCACTCCCACCGTGGTTACTAA SEQ ID NO:10 (Amino acid sequence of En8 - OMP43) MKTSNPGVDLIKGFEGLRLKAYDDGVGVWTIGFGTIKYPNGVRVKKGDTCTESQAEEYLRNDLVVFESAINRLVKVPLNQNQFDALASFTYNLGEGNLSISTLLKKLNAKDYKGAAAEFPKWNKAGGRVL AGLVKRRKAEMELFLKNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKGAKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLAGAGAGKFHEKHHSHRGY Example 2: Shake-flask expression of recombinant protein One transformant was picked from the transformation plate and inoculated into 5 mL of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl). The transformant was incubated at 37 °C with a shaker at 180 rpm for 4 h. Then, 1% of the transformant was inoculated into 50 mL of fermentation medium (2.5% tryptone, 1.25% yeast extract, 0.75% NaCl, 0.6% ammonium chloride, 0.6% lactose). The transformant was incubated overnight (16 h) at 30 °C with a shaker at 200 rpm. Lactose induced the expression of the target protein.

[0043] Take 1 mL of fermentation broth, centrifuge at 12,000 rpm for 1 min, and collect the supernatant.

[0044] Take 80 μL of supernatant, add 20 μL of 5× SDS-PAGE protein loading buffer, and boil for 5 min. Load 20 μL of the sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After staining, destain and analyze the target bands (e.g., ...). Figure 2 ).

[0045] Example 3 Determination of the target protein Total protein content was determined using the Bradford method, and the specific steps are as follows: 1) Plotting the standard curve: Add reagents to the centrifuge tubes according to the dosages in Table 1, then add 1 mL of Bradford working solution (purchased from Shanghai Sangon Biotech Co., Ltd.) to each tube and mix immediately. Let stand at room temperature for 5-10 minutes, then measure the absorbance at 595 nm using a spectrophotometer. Zero the blank. Plot the standard protein content (μg) on ​​the x-axis. Using the ordinate as the vertical axis, fit a linear equation (y = kx + b).

[0046] The fitted standard curve is: y = 0.0088x + 0.0017 (R² = 0.9997) Table 1. Standard curve plotting for total protein determination using the Bradford method, including the dosage of each test tube. Pipe / Hole Number BSA (1 mg / mL) Distilled water / buffer solution Protein content (μg) blank 0 100 0 S1 10 90 10 S2 20 80 20 S3 50 50 50 S4 80 20 80 S5 100 0 100 2) Determination of total protein content in fermentation broth 100 μL of fermentation broth was added to 1 mL of Bradford working solution, and the total protein content in each fermentation broth was determined. The results are shown in Table 2.

[0047] Table 2 Total protein content in each fermentation broth Total protein content (μg) En6 115.4 En6-OMP43 145.6 En8 301.4 En8-OMP43 373.3 The specific steps for determining the content of the target protein in the fermentation broth are as follows: The percentage of the target protein in each lane of the protein electrophoresis image was analyzed using ImageJ software, and this percentage was used as the percentage of the target protein in the total protein. The content of the target protein in each fermentation broth was calculated, and the results are shown in Table 3. The expression levels of En6 and En6-OMP43 were 71.2 μg / mL and 94.5 μg / mL, respectively, with En6-OMP43 showing a 32.7% increase in expression level compared to En6. The expression levels of En8 and En8-OMP43 were 146.2 μg / mL and 198.9 μg / mL, respectively, with En8-OMP43 showing a 36.1% increase in expression level compared to En8. This indicates that protein OMP43 has strong versatility and can increase the expression levels of different target proteins.

[0048] Table 3. Content of target protein in each fermentation broth Percentage of target protein (%) Target protein content (μg / mL) En6 61.7 71.2 En6-OMP43 64.9 94.5 En8 48.5 146.2 En8-OMP43 53.3 198.9 Example 4: In vitro bactericidal activity assay Acinetobacter baumannii (ATCC 19606) cultured overnight was transferred and grown to the mid-log phase (OD). 600 The bacterial cells were collected by centrifugation (5000 rpm, 5 minutes) at approximately 0.5 μL. The cells were washed twice with 20 mM PBS (pH 7.2) and then resuspended in 20 mM PBS buffer. In a 96-well plate, 100 μL of bacterial culture, 50 μL of equimolar fermentation broth, and 1 μL of 200 mM EDTA sample (final concentration 2 μg / mL) were added sequentially. PBS buffer was then added to bring the total reaction volume to 200 μL. The reaction was carried out at 37 ℃, and the readings were taken at 600 nm using a microplate reader.

[0049] The turbidimetric test results are shown below. Figure 3As shown, the bactericidal ability of En6-OMP43 and En8-OMP43 against Acinetobacter baumannii is consistent with that of the corresponding unfused OMP43 En6 and En8, indicating that OMP43 can maintain the original biological activity of the target protein while increasing the expression level.

[0050] Example 5: Determination of minimum inhibitory concentration (MIC) in vitro Acinetobacter baumannii cultured overnight was diluted to 1.0 OD / mL with LB medium, and then further diluted to 10 OD / mL using a tenfold gradient method. 4 CFU / mL, for later use. Transfer 100 μL of test bacterial culture to a 96-well cell culture plate. Dilute the purified lysin antimicrobial protein to 1 mg / mL, then further serially dilute with LB medium. Transfer 100 μL of each dilution of the lysin antimicrobial protein to a 96-well plate, mix thoroughly with the test bacterial culture, and incubate at 37 ℃ for 48 h. Use LB medium as a negative control. Set up three replicates per well.

[0051] The results of the in vitro MIC assay of the lysin antimicrobial proteins are shown in Table 4. En6, En6-OMP43, En8, and En8-OMP43 all showed good antibacterial activity against Acinetobacter baumannii. The MICs of En6 and En6-OMP43 were 4 μg / mL and 2 μg / mL, respectively. The above results indicate that the OMP43 fusion construct can maintain the antibacterial ability of the lysin against Acinetobacter baumannii under the conditions of the embodiments of the present invention. Taking En6 as an example, the recombinant protein En6-OMP43 after fusion with OMP43 has a smaller MIC and stronger antibacterial activity.

[0052] Table 4 Results of in vitro MIC assay Lysing enzyme MIC level (μg / mL) En6 4 En6-OMP43 2 The above embodiments are merely preferred embodiments of the present invention and are only used to explain the present invention, not to limit the present invention. Any changes, substitutions, modifications, etc., made by those skilled in the art without departing from the spirit and essence of the present invention should be within the protection scope of the present invention.

Claims

1. A protein OMP43, characterized in that, Includes any one of the amino acid sequences shown in (1)-(2): (1) An amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO:4 and has the same function; (2) An amino acid sequence with the same function obtained by modifying, substituting, deleting or adding one or more amino acids of the amino acid sequence shown in SEQ ID NO:

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the protein OMP43 as described in claim 1.

3. A recombinant protein, characterized in that, The recombinant protein comprises the target protein and the protein OMP43 as described in claim 1, wherein the protein OMP43 is fused to the target protein.

4. The recombinant protein according to claim 3, characterized in that, The protein OMP43 is fused to the C-terminus of the target protein; and / or, The target protein contains a flexible linker with protein OMP43; and / or, The target protein includes phage lysin En6, the amino acid sequence of which is shown in SEQ ID NO:2, and the encoding nucleotide sequence of which is shown in SEQ ID NO:

1.

5. The recombinant protein according to claim 3, characterized in that, The recombinant protein includes recombinant protein En6-OMP43; The amino acid sequence of the recombinant protein En6-OMP43 is shown in SEQ ID NO:6, and the encoding nucleotide sequence of the recombinant protein En6-OMP43 is shown in SEQ ID NO:

5.

6. A recombinant nucleic acid molecule, characterized in that, The recombinant nucleic acid molecule encodes the recombinant protein according to any one of claims 3-5.

7. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the recombinant nucleic acid molecule of claim 6.

8. A host cell, characterized in that, The host cell comprises the recombinant expression vector of claim 7.

9. The application of the protein OMP43 according to claim 1 in improving the expression level of the target protein.

10. The use of the recombinant protein according to any one of claims 3-5 in the preparation of a medicament for the prevention and / or prevention of Acinetobacter baumannii infection.

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