A promoter and its application in enhancing the expression of meningococcal protein

Abstract

The present invention discloses a promoter and its application in enhancing the expression of meningococcal protein. The nucleotide sequence of the promoter comprises random saturation mutations compared with the nucleotide sequence shown in SEQ ID NO:5, and the promoter has improved promoter activity in meningococci compared with the polynucleotide shown in SEQ ID NO:5, which can be enhanced up to 4 times or more.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and particularly relates to a promoter and application thereof in enhancing the expression of meningococcal protein. Background Art

[0002] Neisseria meningitidis ( Neisseria meningitidis , Nm), also known as meningococcus, is a Gram-negative diplococcus that is mainly transmitted through respiratory droplets. The invasive meningitis it causes has a high mortality rate and the risk of serious sequelae. According to studies, the mortality rate of the disease can reach 15%, and 30% of cases will suffer from long-term serious sequelae, posing a major threat to public health.

[0003] Global epidemiological data show that serogroups A, B, C, W, and Y are the main pathogens causing invasive meningitis, accounting for more than 95% of the cases worldwide. In particular, the prevalence of group B meningococci has been on the rise in recent years, accounting for about 48.5% of global meningococcal cases. In China, the prevalence of group B meningococci is consistent with the global trend and is the highest among all serotypes. This shows that protective measures and vaccine research and development for group B are particularly urgent.

[0004] Internationally, there are two group B meningococcal vaccines that have been successfully marketed, namely the outer membrane vesicle (OMV) vaccine Bexsero and the recombinant protein vaccine Trumenba.

[0005] In the process of meningococcal vaccine development, gene editing technology has become a very important tool and means. However, as a non-model organism, there are still many challenges in using gene editing methods to regulate gene expression in meningococci. Its genomic characteristics and metabolic mechanisms are quite different from those of common model organisms such as Escherichia coli, which makes it difficult for common gene regulation tools, such as promoters and other regulatory elements, to play their advantages in meningococci, which creates many obstacles to the development of meningococcal vaccines. Especially in terms of antigen expression, many commonly used strong promoters have low expression efficiency in meningococci and cannot achieve the ideal antigen expression level.

[0006] At present, the highly active and strong promoters discovered in meningococci can be divided into two categories: inducible promoters and constitutive promoters. Inducible promoters include IPTG (isopropyl-β-D-thiogalactopyranoside)-induced Plac promoter and tetracycline-induced Ptet promoter. [4] The constitutive promoter includes a promoter sequence part-porA obtained by modifying the polyG sequence in the endogenous promoter of the porA gene. [5] , and the promoter PopaB from Neisseria gonorrhoeae [4]. Although the inducible promoter has a high expression level, it requires the addition of specific inducers, which is not conducive to industrial production. In meningococci, if the existing endogenous constitutive strong promoters are used, such as the promoter sequences of the porA or porB genes, although no induction is required, the types are limited. However, due to the limited types of existing known meningococcal endogenous constitutive promoters, it is difficult to meet the needs of simultaneous high expression of multiple antigens. If other exogenous promoters are used, there is often a problem that the expression characteristics of the exogenous promoter are not coordinated with the target bacteria, and it is difficult to achieve the expected effect.

[0007] Therefore, in order to improve the expression efficiency and immunogenicity of meningococcal vaccine antigens, it is crucial to develop new and efficient meningococcal-specific promoter tools. Summary of the invention

[0008] In order to solve the technical problems of insufficient gene regulation tools and low gene expression efficiency of meningococci as a non-model bacterium, the present invention provides a promoter and its application in enhancing the expression of meningococcal proteins. The promoter can significantly improve the expression efficiency and immunogenicity of meningococcal vaccine antigens, laying the foundation for the development of meningococcal vaccines.

[0009] The present invention solves the above technical problems through the following technical solutions.

[0010] A first aspect of the present invention provides a promoter, wherein the nucleotide sequence of the promoter comprises random saturation mutations compared to the nucleotide sequence shown in SEQ ID NO: 5, and the promoter has improved promoter activity compared to the polynucleotide shown in SEQ ID NO: 5.

[0011] In some embodiments of the present invention, the nucleotide sequence of the promoter undergoes random saturation mutation from position 178 to position 194 compared to the nucleotide sequence shown in SEQ ID NO: 5.

[0012] In some embodiments of the present invention, the nucleotide sequence of the promoter is shown in SEQ ID NO: 15, 16 or 17.

[0013] In some embodiments of the present invention, the promoter has improved promoter activity in prokaryotes, preferably Neisseria such as meningococci, compared to the polynucleotide shown in SEQ ID NO: 5.

[0014] The second aspect of the present invention provides an expression cassette, which comprises the promoter as described in the first aspect of the present invention or the promoter whose nucleotide sequence is shown as SEQ ID NO: 5.

[0015] In some embodiments of the present invention, the expression cassette further comprises one or more of an insertion site, a terminator, a Kozak sequence, an enhancer and a post-transcriptional regulatory element of the target gene.

[0016] In some embodiments of the present invention, the expression cassette further comprises a target gene.

[0017] In some embodiments of the present invention, the target gene and the promoter are operably linked.

[0018] In the present invention, the term "operably linked" refers to the juxtaposition of two or more target biological sequences with or without spacers or linkers, so that they are in a relationship that allows them to function in an intended manner. For example, when a polynucleotide encoding a protein is operably linked to a regulatory sequence such as a promoter or enhancer, it means that the polynucleotide sequence encoding the protein is linked in a manner that allows the regulatory sequence to regulate protein expression.

[0019] In some embodiments of the present invention, the target gene is a gene encoding a meningococcal outer membrane protein.

[0020] In some embodiments of the invention, the meningococcal outer membrane protein is selected from the group consisting of fHbp, NHBA, NadA, PorA, PorB, Opc, NspA, NhhA and FetA.

[0021] In some embodiments of the present invention, the meningococcal outer membrane protein is fHbp protein.

[0022] In some embodiments of the present invention, the amino acid sequence of the fHbp protein is shown in SEQ ID NO: 20.

[0023] In some embodiments of the present invention, the nucleotide sequence of the gene encoding the fHbp protein is shown in SEQ ID NO:19.

[0024] The third aspect of the present invention provides a recombinant vector, which comprises the promoter as described in the first aspect of the present invention or the promoter whose nucleotide sequence is shown as SEQ ID NO: 5, or the expression cassette as described in the second aspect of the present invention.

[0025] In some embodiments of the present invention, the recombinant vector further comprises one or more of a replication origin site, an enhancer, a selection marker, a ribosome binding site and polyA.

[0026] In some embodiments of the present invention, the selection marker is one or more of an antibiotic selection marker, a fluorescent protein gene, and an anti-screening gene (eg, rpsL anti-screening gene).

[0027] In some embodiments of the present invention, the antibiotic selection marker is one or more of kanamycin resistance gene (KanR), ampicillin resistance gene, tetracycline resistance gene, chloramphenicol acetyltransferase gene (CmR), streptomycin acetyltransferase gene (Strr), neomycin resistance gene, hygromycin phosphotransferase gene (hph) and gentamicin acetyltransferase gene.

[0028] In some embodiments of the present invention, the recombinant vector is an expression vector or a cloning vector.

[0029] In some embodiments of the present invention, the backbone plasmid of the expression vector is pcDNA3.1, pEGFP, or pET series vectors; the backbone plasmid of the cloning vector is pGEM series vectors, pUC series vectors such as pUC18 / 19, or pMAL-p5X.

[0030] The fourth aspect of the present invention provides a recombinant meningococcal cell, the recombinant meningococcal cell comprising the promoter as described in the first aspect of the present invention or the promoter whose nucleotide sequence is shown in SEQ ID NO: 5, the expression cassette as described in the second aspect of the present invention or the recombinant vector as described in the third aspect of the present invention. In the recombinant meningococcal cell, the promoter drives the expression of the meningococcal outer membrane protein.

[0031] In some embodiments of the invention, the meningococcal cell is derived from Neisseria meningitidis serotype B, such as Neisseria meningitidis MC58.

[0032] The fifth aspect of the present invention provides the use of the promoter as described in the first aspect of the present invention or the promoter with a nucleotide sequence such as SEQ ID NO: 5, or the expression cassette as described in the second aspect of the present invention in preparing a recombinant vector or a recombinant meningococcal cell.

[0033] The fifth aspect of the present invention provides the use of the promoter as described in the first aspect of the present invention or the promoter with a nucleotide sequence as shown in SEQ ID NO: 5, the expression cassette as described in the second aspect of the present invention, the recombinant vector as described in the third aspect of the present invention or the recombinant meningococcal cell as described in the fourth aspect of the present invention in at least one of the following:

[0034] a) enhancing the transcriptional expression level of a gene in meningococcal cells, or preparing a reagent or kit for enhancing the transcriptional expression level of a gene in meningococcal cells;

[0035] b) preparing meningococcal outer membrane proteins, or preparing reagents or kits for preparing meningococcal outer membrane proteins;

[0036] c) producing meningococcal outer membrane vesicles, or producing a reagent or kit for preparing meningococcal outer membrane vesicles; the meningococcal outer membrane vesicles contain the meningococcal outer membrane protein;

[0037] d) Preparation of vaccines or drugs for the prevention or treatment of meningitis.

[0038] The term "outer membrane vesicle" refers to vesicles extruded from the outer membrane (OM) of Gram-negative bacteria, with a size between 20nm and 200nm. Outer membrane vesicles are believed to be formed by Gram-negative bacteria through the process of budding, and budding is believed to be related to the weakening of the connection between the outer membrane and the peptidoglycan layer or the accumulation of substances such as proteins in the periplasmic space. Outer membrane vesicles play a role in the regulation of long-distance delivery, biofilm formation, bacterial survival, and bacterial interactions. On the one hand, outer membrane vesicles are small in size and can be easily absorbed by antigen-presenting cells; on the other hand, outer membrane vesicles have a lot in common with bacteria and are closer to simulating natural infections; on the other hand, outer membrane vesicles contain LPS, which can act as a natural adjuvant. Therefore, outer membrane vesicles are considered to be a new generation of vaccine forms or antigen delivery carriers with great potential.

[0039] In some embodiments of the invention, the meningococcal outer membrane protein is selected from the group consisting of fHbp, NHBA, NadA, PorA, PorB, Opc, NspA, NhhA and FetA.

[0040] In some embodiments of the present invention, the meningococcal outer membrane protein is fHbp protein.

[0041] In some embodiments of the present invention, the amino acid sequence of the fHbp protein is shown in SEQ ID NO: 20.

[0042] The sixth aspect of the present invention provides a method for producing meningococcal outer membrane vesicles or preparing meningococcal outer membrane proteins, the method comprising culturing the recombinant meningococcal cells as described in the fourth aspect of the present invention to obtain the meningococcal outer membrane vesicles or meningococcal outer membrane proteins; the meningococcal outer membrane vesicles contain the meningococcal outer membrane proteins.

[0043] In some embodiments of the invention, the meningococcal outer membrane protein is selected from the group consisting of fHbp, NHBA, NadA, PorA, PorB, Opc, NspA, NhhA and FetA.

[0044] In some embodiments of the present invention, the meningococcal outer membrane protein is fHbp protein.

[0045] In some embodiments of the present invention, the amino acid sequence of the fHbp protein is shown in SEQ ID NO: 20.

[0046] A seventh aspect of the present invention provides a method for constructing a meningococcal promoter mutant library, the method comprising:

[0047] a) constructing a wild-type promoter comprising the polynucleotide sequence shown in SEQ ID NO: 5;

[0048] b) Random saturation mutation was performed on 17 bp from position 178 to position 194 of SEQ ID NO: 5 to obtain a promoter mutant.

[0049] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0050] The reagents and raw materials used in the present invention are commercially available.

[0051] The positive and progressive effects of the present invention are:

[0052] The present invention is based on the wild-type promoter shown in SEQ ID NO: 5, and screens out promoters (such as P5-28C, P5-89C, P5-46) with higher transcriptional expression activity than the wild-type promoter through mutation, and the activity can be enhanced by up to 4 times or more. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A framework map of the pTT-0163 plasmid is shown.

[0054] Figure 2 The fold change of fluorescence intensity under different promoters is shown.

[0055] Figure 3 The P5 promoter mutation region is shown.

[0056] Figure 4 A framework diagram of the mutant library sequence FTT-0832 is shown.

[0057] Figure 5 The fold change in promoter fluorescence for the highly active mutant library is shown.

[0058] Figure 6 Western blot results of FHBP antigen expression are shown. DETAILED DESCRIPTION

[0059] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0060] Different types of promoters have different regulatory mechanisms. When engineering strains, the stability of gene expression is particularly important due to the stability requirements of subsequent production. Therefore, when selecting a promoter, not only its transcription level should be considered, but also its expression stability should be one of the key screening criteria. The expression of essential genes is less affected by environmental factors and is usually continuously expressed at all growth stages of the strain, making them more suitable for engineering bacteria. [2, 3] , 8 promoter sequences of meningococci that start essential genes for growth and have high transcription intensity were selected, and they were named P1 (sequence SEQ ID NO: 1), P2 (SEQ ID NO: 2), P3 (SEQ ID NO: 3), P4 (SEQ ID NO: 4), P5 (SEQ ID NO: 5), P6 (SEQ ID NO: 6), P7 (SEQ ID NO: 7), P8 (SEQ ID NO: 8), and the promoter information of the 8 essential genes is shown in Table 1. In the following embodiments, the reverse screening gene is used to screen strains that have undergone a second homologous recombination. The strain after the second homologous recombination is a strain for achieving seamless gene editing, which does not contain other marker (screening) genes except the target gene. Since Examples 1 and 2 judge the promoter strength by GFP fluorescence intensity, the strain only needs to undergo the first recombination and integrate the exogenous gene (such as GFP) into the genome, and no reverse screening step is required. In Example 3, the effect of introducing only the target gene fHbp is achieved through seamless gene editing, so a reverse screening step is required. For the principle, method and steps of reverse screening using reverse screening genes, please refer to CN117947054B.

[0061] Table 1 Promoters of eight essential genes in meningococci

[0062]

[0063] Example 1 Screening of highly active promoters that highly express other genes from eight endogenous essential genes of meningococci

[0064] The selected promoters are all sequences within 300 bp upstream of the start codon ATG in the 8 essential genes. This example uses sfGFP as a reporter gene, and compares the fluorescence levels of different promoters expressing the same sfGFP in meningococci to screen out endogenous promoters that highly express other genes.

[0065] 1.1 Expression vector plasmid construction

[0066] The expression plasmid vector sequence constructed and used by the present invention comprises a replicon colE1 (positions 1 to 589 in SEQ ID NO: 9), a kanamycin resistance gene (KanR) (positions 3777 to 4592 in SEQ ID NO: 9), an rpsL anti-screening gene (positions 2914 to 3285 in SEQ ID NO: 9), an upstream homology arm sequence U1 (positions 981 to 1780 in SEQ ID NO: 9), a downstream homology arm sequence D1 (positions 4763 to 5562 in SEQ ID NO: 9), a sfGFP green fluorescent protein gene located between the upper and lower homology arms U1 and D1 (positions 1866 to 2603 in SEQ ID NO: 9), and a promoter sequence operably linked to sfGFP and located upstream of the sfGFP green fluorescent protein gene, and further comprises DUS (meningococcal DNA uptake sequence, SEQ ID NO: 9, 5583rd to 5615th).

[0067] In order to screen highly expressed promoters from promoters P1-P8, the present invention constructed expression vector plasmids pTT-0163, pTT-0164, pTT-0165, pTT-0166, pTT-0167, pTT-0168, pTT-0169, and pTT-0170, wherein the promoters are P1, P2, P3, P4, P5, P6, P7, and P8, respectively. The sequence of pTT-0163 is shown in SEQ ID NO: 9, and the expression plasmid vector skeleton thereof is shown in Figure 1 As shown; other plasmids were obtained by replacing the P1 promoter sequence in pTT-0163 with P2-P8 promoters respectively through PCR and recombinase technology.

[0068] 1.2 Strain transformation and verification

[0069] The expression vector plasmids pTT-0163, pTT-0164, pTT-0165, pTT-0166, pTT-0167, pTT-0168, pTT-0169, and pTT-0170 obtained in step 1.1 above were introduced into wild-type meningococcal MC58 by natural transformation, thereby obtaining 8 strains in which the promoter and sfGFP gene were recombined into the genome of MC58. [8]The experimental details are as follows: 1 μg of the above expression vector plasmid was added to the surface of the solid culture medium inoculated with MC58 bacteria, and cultured in a 37°C, 5% CO2 incubator for 8 hours. All bacteria were scraped and spread on a kanamycin-resistant plate, and cultured in a 37°C, 5% CO2 incubator for about 24 hours. The transformed single clones were picked, and the primer F1 (SEQ ID NO: 10) further upstream of the upstream homologous arm and the primer R1 (SEQ ID NO: 11) further downstream of the downstream homologous arm were selected for PCR amplification verification and sent for sequencing verification, and the strains Nme0485 with the sfGFP gene connected to the P1 promoter, the strain Nme0486 with the sfGFP gene connected to the P2 promoter, the strain Nme0487 with the sfGFP gene connected to the P3 promoter, the strain Nme0488 with the sfGFP gene connected to the P4 promoter, the strain Nme0489 with the sfGFP gene connected to the P5 promoter, the strain Nme0490 with the sfGFP gene connected to the P6 promoter, the strain Nme0491 with the sfGFP gene connected to the P7 promoter, and the strain Nme0492 with the sfGFP gene connected to the P8 promoter were screened out.

[0070] 1.3 Fluorescence determination of strains

[0071] The above 8 fluorescent expression strains were inoculated into Tryptic Soy Broth (TSB) liquid culture medium, and the OD of the bacterial solution was measured after 5 hours of culture. 600 The fluorescence intensity of the eight strains was divided by OD 600 The measured values ​​were corrected for bacterial load, and the fluorescence expression was corrected for the background of strain fluorescence. 600 The relative strength of each promoter was determined by the sfGFP fluorescence expression level.

[0072] The results are as follows Figure 2 Among the eight promoters tested, Nme0489 had the strongest fluorescence expression, so the P5 promoter was selected as a highly active endogenous promoter, and the P5 promoter was further mutated and optimized.

[0073] Example 2 Construction of P5 promoter mutation library and screening of highly active mutant promoters

[0074] In order to further enhance the transcription level of the P5 promoter and obtain more promoters with high transcription levels, the present embodiment performs random saturation mutation on a 17 bp sequence near the start site of the P5 promoter region. Figure 3 shown.

[0075] 2.1 Construction of P5 promoter mutant sequence

[0076] In this experiment, the PCR method was used to obtain Figure 4 The DNA fragment library shown contains the upstream homology arm U1 of the insertion site (positions 981 to 1780 in SEQ ID NO: 9), the mutated P5 promoter (as shown in SEQ ID NO: 13), sfGFP (positions 1866 to 2603 in SEQ ID NO: 9), the rpsL anti-screening gene (positions 2914 to 3285 in SEQ ID NO: 9), the chloramphenicol resistance gene (CmR, sequence as shown in SEQ ID NO: 14), the downstream homology arm D3 (positions 4763 to 5392 in SEQ ID NO: 9) and DUS (meningococcal DNA uptake sequence, positions 5583 to 5615 in SEQ ID NO: 9).

[0077] The sequence of the DNA fragment library is shown in SEQ ID NO: 12, which contains a 17 bp mutation region (the same as the N in the mutated P5 promoter of SEQ ID NO: 13), and each base N can be a random one of ATCG.

[0078] The mutated P5 promoter was obtained by designing and synthesizing degenerate primers (N=A+C+G+T) during the PCR process.

[0079] 2.2 Construction of P5 promoter mutant strain library

[0080] The DNA fragment library obtained in 2.1 was transformed into wild-type MC58 via fragments to obtain a series of strain clones containing promoter mutations.

[0081] 2.3 Fluorescence measurement of mutant library strains

[0082] The same method as in Example 1 was used to measure the fluorescence change fold of 228 monoclones in the mutant strain library, and 3 monoclones with enhanced expression were screened out. The above 3 monoclones with enhanced expression were selected, and the promoter region was amplified by PCR and sequenced. It was found that the promoter region sequence of the Nme0493L-28C clone was shown in SEQ ID NO: 15, the promoter region sequence of the Nme0493L-89C clone was shown in SEQ ID NO: 16, and the promoter region sequence of the Nme0493L-46 clone was shown in SEQ ID NO: 17. The fluorescence change fold is shown in Figure 5 As shown, the control strain is Nme0499 with the original P5 promoter and part-porA (sequence shown in SEQ ID NO: 18) constructed from the same batch as the mutant library.[5] Nme0501. From Figure 5 It can be seen that the fluorescence change folds of strains Nme0501, Nme04 93L-28C, Nme0493L-89C, and Nme0493L-46 are 0.49 times, 1.32 times, 1.65 times, and 2.02 times compared to Nme0499 (original P5 promoter), respectively. It is worth noting that compared with the strong promoter part-porA in meningococci reported in previous literature, the expression of the P5 series promoters is enhanced by more than 2 times, and the mutated P5-46 (corresponding to strain Nme0493L-46) can be enhanced by more than 4 times. Resequencing unexpectedly found that in addition to the target mutation region, the site of the P5-46 mutation also found a spontaneous mutation of C→T at the promoter (135bp upstream of ATG) (SEQ ID NO: 17).

[0083] In summary, this example successfully screened and obtained highly active promoters P5-28C, P5-89C, and P5-46.

[0084] Example 3 Screened high-activity promoters for antigen expression

[0085] In order to further determine the expression levels of other proteins such as antigens initiated by the three highly active promoters P5-28C, P5-89C, and P5-46, the key antigen component fHbp protein (nucleic acid sequence: SEQ ID NO: 19, protein sequence: SEQ ID NO: 20) that has been used in the marketed TRUMENBA vaccine product was selected for testing. The three promoters were used to respectively initiate the expression of the MC58 antigen gene fhbp, and the fHbp antigen expression level was determined by Western Blot.

[0086] 3.1 Construction of strains expressing fHbp with different promoters

[0087] a) Construction of a single transformation fragment

[0088] A primary transformation fragment was constructed by fusion PCR, which consisted of an upstream homology arm fragment U2 (SEQ ID NO: 21), a promoter fragment (P5, part-porA, P5-28C, P5-89C or P5-46), an fhbp gene fragment, a chloramphenicol resistance gene (CmR), an rpsL gene, a downstream homology arm fragment D2 (SEQ ID NO: 22) and DUS (meningococcal DNA uptake sequence, positions 5583 to 5615 in SEQ ID NO: 9).

[0089] b) Primary transformation and strain verification

[0090] The first transformation fragment was transferred into the MC58 streptomycin-resistant strain (rpsL (K43R)) by natural transformation, and the strain with successful recombination of the fragment into the genome was selected. The experimental details are as follows: 1 μg of the first transformation fragment was added to the MC58 meningococcus and cultured in a 37°C, 5% CO2 incubator for 8 hours. All the bacteria were scraped and spread on a chloramphenicol-resistant plate and cultured in a 37°C, 5% CO2 incubator for about 24 hours. The single clone grown by transformation was picked, and the upstream homology arm primer F2 (SEQ ID NO: 23) and the downstream homology arm primer R2 (SEQ ID NO: 24) were selected for PCR amplification verification. The original strain amplified a size of about 2.3kbp, and the strain successfully transformed once amplified about 5.3kbp.

[0091] c) Construction of reverse screening transformation fragments

[0092] The reverse screening transformation fragment was constructed by fusion PCR, and the reverse screening transformation fragment consisted of an upstream homology arm fragment U2 (SEQ ID NO: 21), a promoter fragment (P5, part-porA, P5-28C, P5-89C or P5-46), an fhbp gene fragment, a downstream homology arm fragment D2 (SEQ ID NO: 22) and DUS (meningococcal DNA uptake sequence, positions 5583 to 5615 in SEQ ID NO: 9).

[0093] d) Counter-screening transformation and strain verification

[0094] The reverse screening transformation fragment was transferred into the corresponding strain with successful recombination in the first time by natural transformation method, and the strain with successful reverse screening was selected. The experimental details are as follows: 1 μg of reverse screening transformation fragment was added to the corresponding strain with successful recombination in the first time, and cultured in a 37°C, 5% CO2 incubator for 8 hours. All bacteria were scraped and applied to streptomycin resistance plates, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The strain with successful reverse screening lost the streptomycin sensitive gene during the secondary recombination process, so the streptomycin resistance phenotype was restored. The single clone that can grow on the streptomycin resistance plate after transformation was picked, and the primers F2 upstream of the upstream homologous arm and R2 downstream of the downstream homologous arm were selected for PCR amplification verification, and the promoter sequence and fhbp gene sequence were sequenced to verify. The strains Nme0514 (Part-porA promoter), Nme0515 (P5 promoter), Nme0516 (P5-28C promoter), Nme0517 (P5-89C promoter), and Nme0518 (P5-46 promoter) were obtained.

[0095] 3.2 FHbp expression determination

[0096] The FHbp expression of high-activity promoter strains Nme0515, Nme0516, Nme0517, and Nme0518 was detected by Western blot, and MC58 and Nme0514 were used as controls. The experimental details are as follows: Take the lysed bacterial solution and perform polyacrylamide gel electrophoresis (SDS-PAGE). The protein after electrophoresis is transferred to a PVDF membrane (millipore, IEVH85R). After the transfer, the PVDF membrane containing the protein is immersed in 5% skim milk for 2 hours, and then the primary antibody solution human anti-fHbp (1:5000 dilution) is added (primary antibody sequence reference [9] After incubation for 1 hour, the cells were washed five times with 1×TBST (Biyuntian, ST667-1L), and then the secondary antibody solution Goat anti-human IgG (HRP) (1:5000 dilution) was added for incubation for 1 hour, and then washed five times with 1×TBST, and finally developed for imaging.

[0097] FHbp expression results Figure 6 As shown, when the same amount of bacteria was loaded, the grayscale of the FHbp band (35KDa) was compared and it was found that the grayscale of the MC58 band was the smallest, and the grayscale of the bands of Nme0516, Nme0517, and Nme0518 was the strongest, and was higher than that of the control strains Nme0514 and Nme0515.

[0098] The above results show that promoters P5, P5-28C, P5-89C, and P5-46 can all efficiently promote fhbp and can be used for high expression of antigens, and the expression levels are stronger than those of wild-type promoters and reported engineered bacteria promoters.

[0099] Example 4 Strains containing highly active promoters are used to produce outer membrane vesicles (OMVs)

[0100] 4.1 Plate activation of bacterial strains

[0101] Take the frozen glycerol tubes of Neisseria meningitidis Nme0516, Nme0517, Nme0518, Nme0514 and Nme0515 from the -80℃ freezer, spread the bacteria in the glycerol tubes on GC agar medium (OXOID) plates in a biological safety cabinet (Thermo), and culture them in a controlled carbon dioxide incubator (STIK) until an appropriate number of colonies grow on the plates.

[0102] 4.2 Shake flask expansion

[0103] The colonies on the plate were inoculated into shake flasks. TSB medium (BD) was used as the shake flask culture medium. The pH was adjusted to 6.5-8. After sterilization and cooling, glucose was added to a concentration of about 3 g / L. The shake flasks were placed in a constant temperature shaking incubator (Minquan) for 5-6 hours.

[0104] 4.3 Fermentation tank culture

[0105] Add sterilized tryptic soy casein broth (TSB) into the fermenter, control the volume of the culture medium at 40%-80% of the volume of the fermenter, inoculate the strain, maintain the initial OD of the fermentation liquid at 0.05-0.3, control the temperature at 33-39°C, pH 6.5-8, air volume at 0.4-2vvm, dissolved oxygen at 10%-60%, link the rotation speed with the dissolved oxygen, and use the fermenter's feed peristaltic pump to add glucose.

[0106] 4.4 Harvesting OMVs

[0107] After the strain grew to the stationary phase, the fermentation was stopped and OMVs were harvested. NTA detected that the OMV particle size was between 50nm and 100nm, and Western blot detected that FHbp was highly expressed in strains Nme0515, Nme0516, Nme0517, and Nme0518.

[0108] The sequences used in the present invention are as follows, wherein bold indicates the mutation region in the wild-type promoter, and bold+underline indicates the mutation region in other sequences.

[0109] P1 promoter (SEQ ID NO: 1): TTCCTGCCTGTCGATGCCGTCTGAATCCGTTTCAGACGGCATTTTTGTTTTCGGATAAAATAGGCGCGTCCAAAGGAATCTGCCT

[0110] P2 promoter (SEQ ID NO: 2): GTACGGACGGACGGTAGGTTTGACGGCGTGTATTTTAAGGCTTATCGGGAAGACGGGCAATTTTCAGACGGCATACGGACGGTAAATGTTGTGAAAATGCCGTTGTCGGCGGCGGATTGTTTGCTGTGGCGAAAAATGTTATCTTTCAAATGATAACCTTTATCAGAAAACT

[0111] P3 promoter (SEQ ID NO: 3): ATTTGATACAATCCGTGCCGTCTGAAGCCCAAACCGGTTTCAGACGGCATTTTGCACCCGACACTTCAGGATTCGGCACAC

[0112] P4 promoter (SEQ ID NO: 4): ACAGGTGGATTGCCGTTTGACAGGTGAGAAGTATTTTGCCAGCAGCAAGATACTTCTTATATAATGAATAATAATTTATTTAAACCGTCCTCTGAATGGGGCGAGCAGGAGTTTTTGA

[0113] P5 promoter (SEQ ID NO: 5): GGCAGACTCTCAGGTTTACGTTTCCGTCAAACGGCAGATACCGCTTGAAAGCTGATTCAAATAGCGGGCAATTATATCAGCAAAGCCATCAGGGTACATCTTTTCAGACGGCATATGGTTATCCGAACCGCGCAAACTTTGACAAAACAGGCAAATAACAATACAATGCCGAGTTCCGCACAATGGTGTGCGGATATTAACCATAACCCTTATGGAGTTGAGT

[0114] P5 mutant promoter (SEQ ID NO: 13): GGCAGACTCTCAGGTTTACGTTTCCGTCAAACGGCAGATACCGCTTGAAAGCTGATTCAAATAGCGGGCAATTATATCAGCAAAGCCATCAGGGTACATCTTTTCAGACGGCATATGGTTATCCGAACCGCGCAAACTTTGACAAAACAGGCAAATAACAATACAATGCCGAGTTCCNNNNNNNNNNNNNNNNNTATTAACCATAACCCTTATGGAGTTGAGT

[0115] P6 promoter (SEQ ID NO: 6): ACAAAAGTTTTCAGACGGCCTTTCAGGGTCGTCTGAAAAAGTGGTTTCAGAATAAGAATGAAGCAATCGGCATTTAGGCCGTCTGAAATCAAAAGTACCGTTTCCCAATATCGAAAATCCGCCATGCGGTAAAAATACTTCCTTCAAGGAGCAAAA

[0116] P7 promoter (SEQ ID NO: 7): CCCTTCCCTGATATGCCGTCTGAAGCCGCTTCAGACGGCATTTTATCAAGCTCTCCGTCCGCCGCACCCGTGCCGTCCGCATCTTACCGCCCACCCTTCCGGCCGCGCCGTTTTCAATAAAATATTAATTACACGCCACTACAAATTTGCTATAATCCGCCCCGAAAATCTACCCAACCCTCAACAAAGGAACAAACC

[0117] P8 promoter (SEQ ID NO: 8):AAAAACATCCACGCTTATCTGTCTTATACAGCAAGTCTTAAACTCAAACTTATTTGACAAAAAGGTAATGAAAATATTGGATATTTGCTACTTACGCACAAGCAGGTTGGCAATCATCAAAATCATCATTAAAATTTTAAATTCAAATATTGATGGTAAAAGTTTTGGAAATCTGTGCAAACAGTAAAATTCATCTATATCAAATAAGCGGCAAGTAGTTAGATGTAATAGTGGAATGTAACTTTATTTCAGAATACAATACAGGTATTGAAATTTCGTTTCACAGCTTGGAGTATATAC

[0118]

[0119] F1(SEQ ID NO: 10):TCAGGTTGACTTTCGGATAGCGT

[0120] R1(SEQ ID NO: 11):GCTCATTCAGTGTTTCAGCGT

[0121]

[0122] CmR(SEQ ID NO: 14):TGATCGGCACGTAAGAGGTTCCAACTTTCACCATAATGAAATAAGATCACTACCGGGCGTATTTTTTGAGTTATCGAGATTTTCAGGAGCTAAGGAAGCTAAAATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGAACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGATATTACGGCCTTTTTAAAGACCGTAAAGAAAAATAAGCACAAGTTTTATCCGGCCTTTATTCACATTCTTGCCCGCCTGATGAATGCTCATCCGGAATTCCGTATGGCAATGAAAGACGGTGAGCTGGTGATATGGGATAGTGTTCACCCTTGTTACACCGTTTTCCATGAGCAAACTGAAACGTTTTCATCGCTCTGGAGTGAATACCACGACGATTTCCGGCAGTTTCTACACATATATTCGCAAGATGTGGCGTGTTACGGTGAAAACCTGGCCTATTTCCCTAAAGGGTTTATTGAGAATATGTTTTTCGTCTCAGCCAATCCCTGGGTGAGTTTCACCAGTTTTGATTTAAACGTGGCCAATATGGACAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCGACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCATGTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTAATTTTTTTAAGGCAGTTATTGGTGCCCTTAAACGCCTGGTGCTACGCCTGAATAAGTGATAATAAGCGGATGAATGGCAGAAATTCGAAAGCAAATTCGACCCGGTCGTCGGTTCAGGGCAGGGTCGTTAAA

[0123] P5-28C(SEQ ID NO: 15):GGCAGACTCTCAGGTTTACGTCCGTCAAACGGAGATACCGCTTGAAGCTGATTCAATAGCGGGCAATTATCAGCAAAGCCATCAGGGTACATCTTCCAGACGGCAT AGGCACACTTGTGCCAT TATTAACCATAACCCTTATGGAGTTGAGT

[0124] P5-89C(SEQ ID NO: 16):GGCAGACTCTCAGGTTTACGTCCGTCAAACGGAGATACCGCTTGAAGCTGATTCAATAGCGGGCAATTATCAGCAAAGCCATCAGGGTACATCTTTCAGACGGCAT ACTTGGTGTATCCTTTT TATTAACCATAACCCTTATGGAGTTGAGT

[0125] P5-46(SEQ ID NO: 17):GGCAGACTCTCAGGTTTACGTCCGTCAAACGCAGATACCGCTTGAAGCTGATTCAATAGCGGGCAATTATCAGCAAAGCCAAAGCAGGGTACATCTTTTCAGACGAT TATGGCATAGGAATGTT TATTAACCATAACCCTTATGGAGTTGAGT

[0126] Part-porA(SEQ ID NO: 18):ATGGTTTTTTCAACAGGAAGATTATTGAAGACGTATCGGGTGTTTGCCCGATGTTTAGGTTTATCAAATTTACAAAAGGAAGCCGAT

[0127] fhbp nucleic acid sequence (SEQ ID NO: 19): ATGCCGTCTGAACCGCCGTTCGGACGACATTTGATTTTTGCTTCTTTGACCTGCCTCATTGATGCGGTATGCAAAAAAAGATACCATAACCAAAATGTTTATATATTATCTATTCTGCGTATGACTAGGAGTAAACCTGTGAATCGAACTGCCTTCTGCTGCCTTTCTCTGACCACTGCCCTGATTCTGACCGCCTGCAGCAGCGGAGGGGGTGGTGTCGCCGCCGACATCGGTGCGGGGCTTGCCGATGCACTAACCGCACCGCTCGACCATAAAGACAAAGGTTTGCAGTCTTTGACGCTGGATCAGTCCGTCAGGAAAAACGAGAAACTGAAGCTGGCGGCACAAGGTGCGGAAAAAACTTATGGAAACGGTGACAGCCTCAATACGGGCAAATTGAAGAACGACAAGGTCAGCCGTTTCGACTTTATCCGCCAAATCGAAGTGGACGGGCAGCTCATTACCTTGGAGAGTGGAGAGTTCCAAGTATACAAACAAAGCCATTCCGCCTTAACCGCCTTTCAGACCGAGCAAATACAAGATTCGGAGCATTCCGGGAAGATGGTTGCGAAACGCCAGTTCAGAATCGGCGACATAGCGGGCGAACATACATCTTTTGACAAGCTTCCCGAAGGCGGCAGGGCGACATATCGCGGGACGGCGTTCGGTTCAGACGATGCCGGCGGAAAACTGACCTACACCATAGATTTCGCCGCCAAGCAGGGAAACGGCAAAATCGAACATTTGAAATCGCCAGAACTCAATGTCGACCTGGCCGCCGCCGATATCAAGCCGGATGGAAAACGCCATGCCGTCATCAGCGGTTCCGTCCTTTACAACCAAGCCGAGAAAGGCAGTTACTCCCTCGGTATCTTTGGCGGAAAAGCCCAGGAAGTTGCCG

[0128] FHBP Protein Sequence (SEQ ID NO: 20): MPSEPPFGRHLIFASLTCLIDAVCKKRYHNQNVYILSILRMTRSKPVNRTAFCCLSLTTALILTACSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIGLAAKQ

[0129] U2 (SEQ ID NO: 21): CACCAGCCACGGCGCATACAAATTCACCCGTCCGCCCACAGGCGATGTATTACGTATCGACCGCATCAAAGAAATCCACCAAGCCCTGCCCAATACACACATCGTGATGCACGGCTCCAGCTCCGTTCCGCAAGAATGGCTGAAAGTCATCAACGAATACGGCGGCAATATCGGCGAAACCTACGGCGTGCCGGTTGAAGAAATCGTCGAAGGCATCAAACACGGCGTGCGCAAAGTCAACATCGATACCGACTTGCGCCTTGCTTCTACCGGCGCGGTACGCCGCTACCTTGCCGAAAATCCGTCCGACTTTGACCCGCGCAAATACCTGAGCAAAACCATTGAGGCCATGAAGCAAATCTGCCTCGACCGTTATCTTGCGTTTGGCTGCGAAGGTCAGGCAGGCAAAATCAAACCTGTTTCGTTGGAAAAAATGGCAAGCCGTTATGCCAAGGGCGAATTGAACCAAATCGTCAAATAA

[0130] D2 (SEQ ID NO: 22): CCATTGTGAAAATGCCGTCCGAACACGATAATTTACCGTTCGGACGGCATTTTGTATTGCACCGTCCGACGGCATGCCCAAGGGGGGAAATCCCTATTTTCAGGCCAACCGCTATATAATGCCGTCTGAACCAACGAGAGAATGCCATGCAAGCTGATTTTAACCGTCCCGTCCTGGCCGTCGATACCGGTACTTCCCGTTTGTCGCTCGCGCTGCGTGCCGACGGCGAAACCCGTCTGTTCCATCAGGAAGTCGGCAGCCGCCAGTCCGAACTGATTCTGCCGGAAATCCGCACCCTATTCCGCGATGCAGGCATTACCGCCGCCGATTTGGGTGCGGTCGTGTACGCACAGGGTCCCGGCGCGTTTACCGGACTGCGTATCGGCATCGGTGTAGCTCAGGGTTTGGCAACGCCGTTTGATACCCCCTTAATCGGCGTACCCTCGCTCGATGCCGCCGCCTCGCTGCCGCCGCCGCAAAGCTGCATCCTTGCCGCTACGGACGCTCGTATGGGCGAAGTGTTTTATGCATGGTTCGATACGCTGAACTGCCACCGTTTGAGCGATTATCAGGTC

[0131] F2 (SEQ ID NO: 23): GAAGAAGACGGTGTAGGCGCAG

[0132] R2 (SEQ ID NO: 24): CCTTGGCTGCCAATGCGAGAA

[0133] References:

[0134] 1. Guimaraes, J.C., M. Rocha, and A.P. Arkin, Transcript level and sequence determinants of protein abundance and noise in Escherichia coli. Nucleic Acids Res, 2014. 42(8): p. 4791-9.

[0135] 2. Yoshida, K., et al., High throughput optimization of mediumcomposition for Escherichia coli protein expression using deep learning andBayesian optimization. J Biosci Bioeng, 2023. 135(2): p. 127-133.

[0136] 3. Friehs, K., Plasmid copy number and plasmid stability. Adv BiochemEng Biotechnol, 2004. 86: p. 47-82.

[0137] 4. Ramsey, M.E., et al., New complementation constructs for inducibleand constitutive gene expression in Neisseria gonorrhoeae and Neisseriameningitidis. Appl Environ Microbiol, 2012. 78(9): p. 3068-78.

[0138] 5. Sanders, H., et al., FetA Antibodies Induced by an Outer MembraneVesicle Vaccine Derived from a Serogroup B Meningococcal Isolate withConstitutive FetA Expression. PLoS One, 2015. 10(10): p. e0140345.

[0139] 6. Ampattu, B.J., et al., Transcriptomic buffering of cryptic geneticvariation contributes to meningococcal virulence. BMC Genomics, 2017. 18(1):p. 282.

[0140] 7. Muir, A., et al., Construction of a complete set of Neisseriameningitidis mutants and its use for the phenotypic profiling of this humanpathogen. Nat Commun, 2020. 11(1): p. 5541.

[0141] 8. van Dam, V. and MP Bos, Generating knock-out and complementationstrains of Neisseria meningitidis. Methods Mol Biol, 2012. 799: p. 55-72.

[0142] 9. López-Sagaseta, J., et al., Crystal structure reveals vaccineelicited bactericidal human antibody targeting a conserved epitope onmeningococcal fHbp. Nat Commun, 2018. 9(1): p. 528.

[0143] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A promoter, characterized in that The nucleotide sequence of the promoter undergoes random saturation mutations from position 178 to position 194 compared to the nucleotide sequence shown in SEQ ID NO: 5, and the promoter has improved promoter activity compared to the polynucleotide shown in SEQ ID NO: 5; the nucleotide sequence of the promoter is shown in SEQ ID NO: 15, 16 or 17.

2. The promoter according to claim 1, characterized in that The promoter has improved promoter activity in meningococci compared to the polynucleotide shown in SEQ ID NO:

5.

3. An expression cassette, characterized in that The expression cassette comprises the promoter according to claim 1 or 2.

4. The expression cassette according to claim 3, characterized in that The expression cassette further comprises one or more of a target gene, a terminator, a Kozak sequence, an enhancer and a post-transcriptional regulatory element; the target gene and the promoter are operably connected.

5. The expression cassette according to claim 4, characterized in that The target gene is a gene encoding meningococcal outer membrane protein.

6. The expression cassette according to claim 5, characterized in that The meningococcal outer membrane protein is selected from the group consisting of fHbp, NHBA, NadA, PorA, PorB, Opc, NspA, NhhA and FetA.

7. The expression cassette according to claim 6, characterized in that The meningococcal outer membrane protein is fHbp protein, and its amino acid sequence is shown in SEQ ID NO:

20.

8. A recombinant vector, characterized in that: The recombinant vector comprises the expression cassette according to any one of claims 3 to 7.

9. The recombinant vector according to claim 8, characterized in that The recombinant vector further comprises one or more of a replication initiation site, an enhancer, a selection marker, a ribosome binding site and polyA.

10. The recombinant vector according to claim 9, characterized in that The screening marker is one or more of an antibiotic screening marker, a fluorescent protein gene and an anti-screening gene.

11. A recombinant meningococcal cell, characterized in that The recombinant meningococcal cell comprises the promoter according to claim 1 or 2, the expression cassette according to any one of claims 3-7 or the recombinant vector according to any one of claims 8-10.

12. The recombinant meningococcal cell of claim 11, wherein The meningococcal cells are derived from Neisseria meningitidis serotype B.

13. The recombinant meningococcal cell of claim 12, wherein The meningococcal cell is Neisseria meningitidis MC58.

14. Use of the promoter according to claim 1 or 2 or the expression cassette according to any one of claims 3 to 7 in preparing a recombinant vector or a recombinant meningococcal cell.

15. Use of the promoter according to claim 1 or 2, the expression cassette according to any one of claims 3 to 7, the recombinant vector according to any one of claims 8 to 10, or the recombinant meningococcal cell according to any one of claims 11 to 13 in at least one of the following: a) enhancing the transcriptional expression level of a gene in meningococcal cells, or preparing a reagent or kit for enhancing the transcriptional expression level of a gene in meningococcal cells; b) preparing meningococcal outer membrane proteins, or preparing reagents or kits for preparing meningococcal outer membrane proteins; c) producing meningococcal outer membrane vesicles, or producing a reagent or kit for preparing meningococcal outer membrane vesicles; the meningococcal outer membrane vesicles contain the meningococcal outer membrane protein.

16. A method for producing meningococcal outer membrane vesicles or outer membrane proteins, characterized in that: The method comprises culturing the recombinant meningococcal cell according to any one of claims 11 to 13, thereby obtaining the meningococcal outer membrane vesicles or meningococcal outer membrane proteins; the meningococcal outer membrane vesicles contain the meningococcal outer membrane proteins.

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