Nucleic acid constructs, vectors and host cells of CRM197

Abstract

The invention belongs to the field of biological medicine, and relates to a nucleic acid construct of CRM197, a vector containing the nucleic acid construct and a host cell. Specifically, the invention relates to an expression vector capable of being stably replicated in corynebacterium diphtheriae and a corynebacterium diphtheriae strain carrying the vector. Specifically, the nucleic acid construct comprises the following elements: a corynebacterium replicase gene (repA), a positive replication regulator gene (per), an Escherichia coli DNA (Deoxyribose Nucleic Acid) replication starting point and a nucleic acid sequence for coding CRM197 protein. The nucleic acid construct or the carrier can effectively improve the yield of CRM197 carrier protein, the purification process of the target protein is simplified, the production efficiency is improved, the production cost is saved, and the nucleic acid construct or the carrier has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a nucleic acid construct of CRM197, a vector and a host cell comprising the nucleic acid construct. Specifically, the present invention relates to an expression vector that can stably replicate in Corynebacterium diphtheriae and a Corynebacterium diphtheriae strain carrying the vector. Background Art

[0002] CRM197 (Cross-reacting forms of toxin 197, also known as Cross Reacting Material 197) is a mutant of diphtheria toxin (DT) with a molecular weight of about 58kD produced by Corynebacterium diphtheriae. Due to the mutation of glycine (Gly) at position 52 of diphtheria toxin to glutamic acid (Glu), the ADP-ribosyltransferase activity and cytotoxicity of diphtheria toxin are lost, but the complete DT structure and immunogenicity are still maintained, and it has broad application prospects as a carrier protein.

[0003] At present, the mainstream method for preparing CRM197 on an industrial scale is fermentation of Corynebacterium diphtheriae. CRM197 accumulates extracellularly in the form of secretory expression. Since the content of other impurity proteins secreted by the host to the extracellular space is extremely low, a high-purity CRM197 protein can be obtained through a simple purification step. Since the yield of CRM197 produced by fermentation of Corynebacterium diphtheriae is low and far from meeting market demand, researchers have tried to increase the fermentation yield of CRM197 through culture medium formulation, fermentation conditions, and genetic engineering in recent years.

[0004] In addition, although the heterologous expression of recombinant CRM197 has been tested in a variety of heterologous hosts (such as Escherichia coli, Bacillus subtilis and Pseudomonas fluorescens, etc.) in recent years, the expression forms are mostly inclusion bodies or intracellular expression, which poses extremely high challenges to the downstream purification process and personnel operation stability. Therefore, the production engineering strains of CRM197 currently widely used in vaccine fermentation production are still mainly Corynebacterium diphtheriae.

[0005] At present, new technical means to increase the production of CRM197 need to be developed. Summary of the invention

[0006] After in-depth research and creative work, the inventors have constructed a new CRM197 expression vector, which can stably replicate in Corynebacterium diphtheriae and effectively increase the yield of CRM197. The present invention also obtains a new per protein and its coding sequence. The following invention is provided:

[0007] One aspect of the present invention relates to a nucleic acid construct comprising the following elements:

[0008] Corynebacterium replicase gene (repA), positive replication regulator gene (per), Escherichia coli DNA replication origin, and sequence encoding CRM197 protein.

[0009] Without being bound by theory, the plasmid designed and constructed by the present invention carries an E. coli replication origin, and can be prepared by rapid amplification of E. coli to conveniently obtain a high-purity plasmid. At the same time, the plasmid can be introduced into diphtheriae Corynebacterium by an electroporation transformation method established by the inventor. Since the plasmid also carries a replication protease gene of a coryneform bacillus, such as diphtheriae Corynebacterium, it can be stably replicated during the passage of the strain.

[0010] In some embodiments of the present invention, the nucleic acid construct further comprises a gene encoding a selection marker;

[0011] Preferably, the gene encoding the selection marker is located between the positive replication regulator gene and the E. coli DNA replication origin, or between the E. coli DNA replication origin and the nucleic acid sequence encoding the CRM197 protein;

[0012] Preferably, the reading frame direction of the gene encoding the selection marker is opposite to the reading frame direction of the sequence encoding the CRM197 protein.

[0013] Without being limited by theory, the gene encoding the selection marker and the gene of CRM197 are in opposite directions, which is beneficial to reduce the probability of read-through.

[0014] In some embodiments of the present invention, the nucleic acid construct further comprises a sequence encoding a signal peptide;

[0015] Preferably, the sequence encoding the signal peptide is linked to the 5' end of the sequence encoding the CRM197 protein.

[0016] Without being bound by theory, the 5' end of the gene sequence of CRM197 on the plasmid contains a DNA sequence encoding a signal peptide, which allows CRM197 to be secreted outside the cell after expression, thereby increasing the yield of the target protein and simplifying the downstream purification process steps, improving production efficiency and saving production costs.

[0017] In some embodiments of the present invention, the nucleic acid construct comprises the following elements in order (e.g., from the 5' end to the 3' end):

[0018] Corynebacterium replicase gene, positive replication regulator gene, gene encoding selection marker, Escherichia coli DNA replication origin, sequence encoding signal peptide, and sequence encoding CRM197 protein;

[0019] Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, gene encoding selection marker, sequence encoding signal peptide, and sequence encoding CRM197 protein;

[0020] Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, antibiotic resistance gene, sequence encoding signal peptide, and DNA sequence encoding CRM197 protein;

[0021] Corynebacterium replicase gene, positive replication regulator gene, antibiotic resistance gene, Escherichia coli DNA replication origin, signal peptide encoding sequence and CRM197 protein encoding DNA sequence; or

[0022] Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, kanamycin resistance gene, sequence encoding signal peptide and DNA sequence encoding CRM197 protein;

[0023] Preferably, the reading frame direction of the gene encoding the selection marker (antibiotic resistance gene such as kanamycin resistance gene) is opposite to the reading frame direction of the sequence encoding the CRM197 protein.

[0024] In some embodiments of the present invention, the nucleic acid construct, wherein the Corynebacterium replicase gene is a Corynebacterium diphtheriae replicase gene; preferably, the Corynebacterium replicase gene encodes the amino acid sequence shown in SEQ ID NO:6; preferably, the Corynebacterium replicase gene comprises the sequence shown in SEQ ID NO:9 or SEQ ID NO:10.

[0025] Without being bound by theory, the CDS region of the replicase gene on the plasmid usually contains elements regulating gene expression within 150-180 bp upstream and downstream, such as promoter, reverse transcribed RNA, Rho-independent transcriptional terminator, etc., which are necessary conditions for the normal translation and function of the target gene. (Khan, SA 1997. Rolling-circle replication of bacterial plasmids. Microbiol. Mol. Biol. Rev. 61: 442-455.).

[0026] In some embodiments of the present invention, the nucleic acid construct, wherein the positive replication regulatory factor gene encodes the amino acid sequence shown in SEQ ID NO:7; preferably, the positive replication regulatory factor gene comprises the sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

[0027] In some embodiments of the present invention, in the nucleic acid construct, the Escherichia coli DNA replication origin comprises the sequence shown in SEQ ID NO:13.

[0028] In some embodiments of the present invention, the nucleic acid construct, wherein the amino acid sequence of the CRM197 protein is as shown in SEQ ID NO:8; preferably, the DNA sequence encoding the CRM197 protein is as shown in SEQ ID NO:16 or SEQ ID NO:3.

[0029] In some embodiments of the present invention, in the nucleic acid construct, the gene encoding the selection marker is an antibiotic resistance gene; preferably, the antibiotic resistance gene is selected from one or more of an ampicillin resistance gene, a chloramphenicol resistance gene, a tetracycline resistance gene and a kanamycin resistance gene.

[0030] In some embodiments of the present invention, the nucleic acid construct, wherein the amino acid sequence of the signal peptide is as shown in SEQ ID NO:20; preferably, the sequence encoding the signal peptide is as shown in SEQ ID NO:21.

[0031] In some embodiments of the present invention, the nucleic acid construct comprises the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in sequence;

[0032] Preferably, the reading frame direction of the kanamycin resistance gene is opposite to the reading frame direction of the sequence encoding the CRM197 protein;

[0033] Preferably, the sequence of the nucleic acid construct is as shown in SEQ ID NO:16.

[0034] Another aspect of the present invention relates to a vector, which comprises the nucleic acid construct described in any one of the present invention; preferably, the vector is a plasmid vector; preferably, the vector is a plasmid expression vector.

[0035] Another aspect of the present invention relates to a recombinant host cell, which comprises the nucleic acid construct or the recombinant vector of any one of the present invention; preferably, the host cell is a recombinant Escherichia coli cell or a Corynebacterium cell such as a recombinant Corynebacterium diphtheriae cell; preferably, a recombinant Corynebacterium diphtheriae ATCC 39255 cell.

[0036] The present invention also relates to a method for constructing engineered diphtheria Corynebacterium, comprising the step of introducing the recombinant vector of the present invention into diphtheria Corynebacterium by using an electroporation method.

[0037] Another aspect of the present invention relates to a cell culture, which comprises the recombinant host cell of the present invention, or is obtained by fermentation culture of the recombinant host cell of the present invention.

[0038] Another aspect of the present invention relates to a method for preparing CRM197 protein, comprising the steps of culturing the recombinant host cell of the present invention under suitable conditions, and isolating and purifying the CRM197 protein; preferably, isolating and purifying the CRM197 protein from the cell culture supernatant.

[0039] In some embodiments of the present invention, the method for preparing CRM197 protein comprises the following steps:

[0040] (1) preparing the original strain of Corynebacterium diphtheriae into competent cells, and introducing the plasmid vector of the present invention into the host Corynebacterium diphtheriae by electroporation transformation, wherein the host Corynebacterium diphtheriae is preferably numbered as ATCC 39255;

[0041] (2) Using YC medium containing antibiotics, screening and obtaining candidate monoclonal clones with resistance, using polymerase chain reaction (PCR) technology to confirm whether the candidate monoclonal clones carry the target plasmid vector, and obtaining the engineered strain of Corynebacterium diphtheriae carrying the target plasmid;

[0042] (3) Using an engineered strain of Corynebacterium diphtheriae carrying the target plasmid to prepare a fermentation seed solution, the fermentation was carried out in a fed-batch manner, the bacterial cells were removed, and the CRM197 protein was harvested and purified from the culture supernatant.

[0043] Another aspect of the present invention relates to an isolated protein comprising the amino acid sequence shown in SEQ ID NO:7.

[0044] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:7; preferably, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

[0045] The term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more operably linked regulatory sequences.

[0046] In the present invention, the term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" can be achieved by means of gene recombination.

[0047] In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide that inhibits a certain protein can be inserted. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). A vector may contain multiple elements for controlling expression.

[0048] In the present invention, the term "host cell" refers to a cell into which a vector is introduced, including many cell types, such as prokaryotic cells such as Escherichia coli or Corynebacterium diphtheriae, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0049] Advantageous Effects of the Invention

[0050] The present invention achieves one or more of the following technical effects:

[0051] (1) The vector or plasmid of the present invention can replicate rapidly.

[0052] (2) The vector or plasmid of the present invention is capable of stably replicating or inheriting.

[0053] (3) The nucleic acid construct, vector or host cell of the present invention can effectively increase the production or yield of CRM197 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 :Sequence alignment of the per protein of the present invention (SEQ ID NO: 7) and the known per protein (SEQ ID NO: 17). The upper sequence is the per protein of the present invention, and the lower sequence is the known per protein.

[0055] Figure 2A : Structural prediction of the per protein (SEQ ID NO: 7) of the present invention.

[0056] Figure 2B: Structural prediction of the known per protein (SEQ ID NO: 17).

[0057] Figure 3 : Optimization results of the coding sequence of CRM197 protein. The left side of the figure is the wild-type coding sequence (SEQ ID NO: 18), and the right side is the optimized coding sequence (SEQ ID NO: 15).

[0058] Figure 4 : Schematic diagram of the construction of the plasmid vector pYHP02.1 that stably replicates in Corynebacterium diphtheriae.

[0059] Figure 5 : Enzyme digestion identification map of plasmid vector pYHP02.1. Lane 1 is the plasmid before digestion, lane 2 is the plasmid after double digestion of pYHP02.1 with restriction endonucleases EcoRI and NcoI, and lane 3 is KB Ladder.

[0060] Figure 6 : PCR screening results of candidate clones of Corynebacterium diphtheriae carrying pYHP02.1 (host ATCC 39255 Corynebacterium diphtheriae). Lanes 1-3 are single clones of Corynebacterium diphtheriae, lane 4 is the original strain without plasmid, and lane 5 is 1KB ladder.

[0061] Figure 7 : Gram staining results of the engineered strain of Corynebacterium diphtheriae carrying the plasmid vector pYHP02.1.

[0062] Figure 8 : SDS-PAGE profile of CRM197 protein fermented by engineered strains of Corynebacterium diphtheriae carrying plasmid vector pYHP02.1. Lane 1 is the supernatant sampled after 10 hours of fermentation of engineered bacteria carrying pYHP02.1 plasmid, lane 2 is a protein marker, lanes 3-7 are the supernatants sampled at different time periods of fermentation of engineered bacteria carrying pYHP02.1 plasmid (12, 13.5, 15, 16.5, 21.6 hours respectively), and lanes 5-6 are the supernatants sampled at different time periods of fermentation of wild-type Corynebacterium diphtheriae (ATCC 39255) (10, 12, 13.5, 15, 16.5, 21.6 hours respectively).

[0063] Fig. 9 : SDS-PAGE gel electrophoresis of the purified CRM197 protein, wherein lanes 1 and 4 are protein markers, lanes 2 and 5 are CRM197 proteins obtained by fermentation purification, and lanes 3 and 6 are standard CRM197 proteins.

[0064] The partial sequence information involved in the present invention is as follows.

[0065] Table A: Partial sequences of the present invention

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[0087] DETAILED DESCRIPTION

[0088] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0089] The Corynebacterium diphtheriae involved in the technical solution in the embodiments described herein was purchased from the American Type Culture Collection (ATCC), and the strain number is ATCC 39255.

[0090] Example 1: Construction of plasmid pYHP02.1 capable of stably replicating in Corynebacterium diphtheriae

[0091] Design a plasmid vector that can stably replicate in Corynebacterium diphtheriae. The plasmid vector is composed of the following three parts:

[0092] (1) The minimal DNA sequence encoding the Corynebacterium replicase protein gene (repA) and the positive effector of replication gene (per) as shown in SEQ ID NO: 1;

[0093] (2) a DNA sequence containing an Escherichia coli replication origin and a kanamycin resistance gene as shown in SEQ ID NO: 2; and

[0094] (3) The DNA sequence encoding the CRM197 protein shown in SEQ ID NO:3.

[0095] To ensure that the replicase can initiate translation in the host, the inventors cut off the DNA sequence of the 155 bp sequence in front of the 5' end of the repA coding sequence (CDS) and the 165 bp sequence in the back of the 3' end.

[0096] The inventors have discovered a DNA sequence that has no homology with the per gene (Positive effector of replication). Although the length of the protein sequence encoded by it is the same as that of the per protein (86 amino acids), the similarity of its amino acid sequence is only 24% ( Figure 1 ).

[0097] The inventors used the de novo folding method to obtain the gene structure of the per gene expression product (https: / / alphafoldserver.com / fold / ). After calculation, the expression product of this gene has a very high consistency with the expression product structure of the per gene (RMSD after superposition of the protein three-dimensional structure = 0.73) ( Figure 2A and Figure 2B ), indicating that the gene can express a protein with similar functions to per protein in diphtheriae Corynebacterium, and may help plasmids to replicate stably. In addition, the inventors also intercepted the 13bp sequence in front of the 5' end of the per coding region and the 26bp DNA sequence in the back of the 3' end as the redundancy of the gene sequence to avoid the phenomenon of gene read-through in the plasmid. Finally, the sequence SEQ ID NO: 1 that can allow plasmids to be stably propagated and replicated in diphtheriae Corynebacterium was obtained.

[0098] SEQ ID NO: 2 contains the kanamycin resistance gene DNA sequence and the E. coli high copy number ColE1 / pMB1 / pBR322 / pUC replication origin to ensure that the E. coli replication origin and the kanamycin resistance gene can function normally in the host.

[0099] SEQ ID NO:3 contains the complete sequence encoding CRM197 protein, and mutates glycine (Gly, sequence GGG) at 52 in the diphtheria toxin coding region to glutamic acid (Glu, sequence GAA, GAG is also acceptable). During protein translation, the formation rate and yield of protein polypeptide chains are highly dependent on the stable secondary structure formed by nucleotide pairing in the mRNA chain. Minimum free energy (MFE) is an important parameter reflecting RNA secondary structure. Currently, the secondary structure of RNA can accurately calculate the MFE generated by nucleotide chain folding by analyzing possible stem-loop structures (http: / / rna.tbi.univie.ac.at / RNAfold). The inventors optimized the coding region sequence by analyzing the RNA sequence and MFE of the CRM197 coding region and combining the codon preference of Corynebacterium diphtheriae (https: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=1717), so that the coding region forms a more stable secondary structure, and the MFE is reduced by more than 30% ( Figure 3 ). Since the upstream and downstream of the coding region usually contain gene structures such as ribosome binding site (RBS), promoter and operator, in order not to affect the normal expression of CRM197, the non-coding region sequences of 236bp upstream and 23bp downstream of the coding region were retained during sequence extraction.

[0100] After the theoretical sequence of plasmid vector pYHP02.1 is designed ( Figure 4 ), and sequentially connected according to SEQ ID NO: 1-3, and artificial synthesis and cyclization reaction were performed to obtain the target plasmid.

[0101] Plasmid vector pYHP02.1 can transform E. coli competent cells treated with CaCl2. E. coli DH5α is used as competent cells. Take 1 tube of competent cells (100 μL / tube) and melt it on ice, then add 0.5-50 ng of plasmid under sterile conditions. After gently mixing, transfer to a 42°C water bath and heat shock for 90 seconds after ice bath for 30 minutes. Quickly transfer the microcentrifuge tube containing competent cells to an ice bath for 5 minutes. Add 600 μL of liquid recovery medium to the tube and incubate in a 37°C water bath for 45 minutes to recover the bacteria. Finally, spread an appropriate amount of bacterial solution on a 50 μg / mL kanamycin resistance plate, invert the plate and culture at 37°C for 12-16 hours.

[0102] When the positive E. coli clones containing the pYHP02.1 plasmid grow out, pick the clones for small-scale culture of 10-15 mL, and use the culture medium to extract a small amount of plasmid. Use restriction endonucleases EcoRI and NcoI to double-digest the obtained plasmid, separate the digestion products by agarose electrophoresis, and analyze the size of the plasmid digestion bands by ultraviolet transmission. After comparing with the theoretical digestion map, determine whether the plasmid is correct. The digestion identification map of pYHP02.1 plasmid is as follows Figure 5 The results showed that the map of pYHP02.1 after double digestion with restriction endonucleases EcoRI and NcoI was consistent with the theoretical calculation. In order to further verify the stability of the plasmid after replication and amplification in E. coli, the key regions of the plasmid with correct digestion were further sequenced and verified, and the nucleotide sequences obtained by sequencing were consistent with the theoretical sequences.

[0103] Example 2: Construction of an engineered strain of Corynebacterium diphtheriae containing the pYHP02.1 plasmid

[0104] The inventors provide a technical solution for introducing plasmid pYHP02.1 into diphtheriae Corynebacterium by electroporation transformation. The specific operation steps are as follows:

[0105] (1) Prepare competent cells of Corynebacterium diphtheriae. Take the frozen strain of Corynebacterium diphtheriae in a glycerol tube (strain number ATCC39255), inoculate it into 2-10 mL of liquid YC medium at an inoculation ratio of 0.25%, and culture it at 37°C, 220 rpm overnight. Transfer 1% of the overnight culture into fresh YC medium, culture it at 37°C, 220 rpm until the OD600 value of the bacterial solution is in the range of 0.4-0.6, and stop the culture (usually the culture time is 2.5-3 hours). Precool the bacterial solution on ice for 20-30 minutes, centrifuge the bacterial solution at 4°C (4000 rpm, 15 minutes), discard the supernatant, add an appropriate volume of buffer 1 (1 mM Tris-HCl, pH 7.5, 10% glycerol) to resuspend the bacterial cells, centrifuge the bacterial solution at 4°C (4000 rpm, 15 minutes), discard the supernatant, and repeat this operation 3 times. Use buffer 2 (10% glycerol) again and repeat the above operation 3 times. Finally, the bacteria were resuspended in 2% culture volume of buffer 2, and the diphtheriae competent cells were dispensed into microcentrifuge tubes (100 μL / tube).

[0106] (2) Electroporation transformation: add 1-2 μg of pYHP02.1 plasmid to the diphtheriae competent cells prepared in (1) and place in an ice bath for 10 min. Transfer the competent cells mixed with the plasmid to an electroporation cup (2 mm) and set the electroporation transformation conditions: voltage 3000 V, 600 Ω, 25 μF, and electric shock time 10-20 ms. Add 1 ml of YC liquid culture to the competent cells after the click is completed, and incubate in a 37°C water bath for 2 hours to allow the bacteria to recover. Finally, spread an appropriate amount of bacterial liquid on a YC medium solid plate (containing 25 μg / mL of kanamycin), invert the plate and culture at 37°C for 24-36 hours.

[0107] (3) Clone screening: When a single clone of Corynebacterium diphtheriae grows out, select candidate clones and inoculate them into a 50 mL sterile bioreactor filled with 5-10 mL YC liquid medium. Culture them on a small scale at 37°C, 220 rpm in a constant temperature shaker for 8-16 hours. Take the bacterial solution and use PCR amplification to verify whether the plasmid has been introduced into the Corynebacterium diphtheriae host. The primer sequences for the PCR reaction are SEQ ID NO: 4 and SEQ ID NO: 5. The gel electrophoresis pattern is shown in Figure 6 shown.

[0108] At the same time, the Gram staining method is used to test the bacterial solution for Corynebacterium diphtheriae. The specific steps are as follows: In the clean bench, pick a small amount of bacteria and spread it evenly on a clean glass slide. Heat it with a flame to make the bacteria adhere to and fix on the glass slide. Stain it with ammonium oxalate crystal violet solution for 1 minute, and remove the floating color with pure water. Stain it with iodine-potassium iodide solution for 1 minute, and decolorize it with 95% ethanol for 30 seconds. Corynebacterium diphtheriae will not fade under a microscope and appear as purple rod-shaped or rod-shaped particles ( Figure 7 ).

[0109] The experiment confirmed that the engineered strain of Corynebacterium diphtheriae containing the pYHP02.1 plasmid could be obtained by electroporation transformation.

[0110] Example 3: Genetic stability experiment

[0111] After obtaining the engineered strain of Corynebacterium diphtheriae containing the pYHP02.1 plasmid, three single clones were selected and continuously subcultured for 10 generations using YC liquid culture medium (inoculation ratio 0.1%, v / v, 220rpm, 37°C, 12h for 1 generation). The subculture of each single clone was taken, and after gradient dilution, it was spread on the YC non-antibiotic solid plate, and single colonies were obtained after overnight culture at 37°C. The single colonies on the plate were picked and inoculated in parallel on the non-antibiotic YC solid plate and the resistant plate (containing kanamycin 25μg / mL), and 100 colonies were inoculated on each plate. After inoculation, the plate was placed in a 37°C incubator for overnight culture, and the number of single colonies on the non-antibiotic YC plate and the number of single colonies on the resistant plate were counted. The plasmid loss rate was calculated according to the following formula: plasmid loss rate = (AB) / Ax100%. Among them, A is the number of single colonies on the non-antibiotic YC plate, and B is the number of single colonies on the resistant plate.

[0112] The results showed that the plasmid loss rates of the three single clones at P10 were 0.8%, 0.5% and 0.7%, respectively, indicating that plasmid pYHP02.1 can stably replicate and propagate in Corynebacterium diphtheriae.

[0113] Example 4: Fermentation of CRM197 using an engineered strain of Corynebacterium diphtheriae carrying the pYHP02.1 plasmid

[0114] The verified engineered strain of Corynebacterium diphtheriae containing the pYHP02.1 plasmid was preserved as a glycerol strain (1 mL / tube, glycerol final concentration 10%), and a cell bank was established. The glycerol strain was inoculated into 500 mL of liquid culture medium (YC culture medium, containing 25 μg / mL kanamycin) at a ratio of 5% to prepare a fermentation seed liquid, and was cultured in a small scale at 37° C. and 220 rpm constant temperature shaker until OD600 reached 3-5, and then transferred to 50 L of fermentation medium, and the fermentation conditions were: 35° C., aeration speed of 10 L / min, stirring speed of 500 rpm, pH 7.2, and a fermentation tank pressure not exceeding 0.03 MPa. The formula of YC medium is: yeast powder (20g / L), glucose (15g / L), potassium dihydrogen phosphate (5g / L), tryptone (10g / L), L-tryptophan (0.05g / L), magnesium sulfate (0.45g / L), calcium chloride dihydrate (0.15g / L), copper sulfate (0.01g / L), zinc sulfate (0.004g / L), L-cystine (0.2g / L), β-alanine (0.004g / L), niacin (0.004g / L), pimelic acid (0.001g / L), manganese chloride tetrahydrate (0.002g / L), and the pH is adjusted to 7.4 after constant volume.

[0115] When the OD600 was 100±10, the fermentation was terminated and the bacterial solution was collected. The bacterial cells were removed by centrifugation, and the expression level of CRM197 in the supernatant was detected by SDS-PAGE gel electrophoresis. The results were as follows: Figure 8 shown.

[0116] The supernatant after centrifugation was filtered through a filter (0.45+0.22μm), and then concentrated to 2L using tangential flow filtration (30KD). The concentrate was further subjected to diafiltration to replace the buffer system with ion exchange chromatography buffer A (20mM, 70mMNaCl). The obtained feed solution was loaded through an injection pump, and the target protein was purified using ion exchange chromatography (DEAE). After injection, the impurities were first washed out using ion exchange chromatography buffer A, and then the target protein CRM197 was eluted using a gradient increasing NaCl concentration. Compared with the original diphtheria Corynebacterium strain, under the same fermentation and purification conditions, the protein yield of CRM197 obtained by fermentation and purification of the diphtheria Corynebacterium engineered strain carrying plasmid pYHP02.1 increased from about 100mg / L to about 165mg / L, an increase of 65%.

[0117] Example 5: Identification of CRM197 protein after purification

[0118] In order to prove that the antigenic epitope of the CRM197 protein obtained by fermentation and purification of the engineered strain of Corynebacterium diphtheriae carrying plasmid pYHP02.1 has not changed, the purified CRM197 protein was separated by SDS-PAGE gel together with the reference substance CRM197 (SERUM INSTITUTE OF INDIA PVT.LTD., Insp lot NO:040000435945), and the protein immunoblotting (Western Blot) method was used to verify whether the purified CRM197 can bind to the diphtheria toxin-specific antibody.

[0119] The experimental method is as follows: about 100 μg of CRM197 protein, standard CRM197 protein and 10 μL molecular weight marker were loaded into the SDS-PAGE gel well. The gel was run at 100 V for 1.5 hours, and the separated proteins on the gel were transferred to PVDF by wet transfer. The membrane was blocked with blocking buffer (5% skim milk powder) at room temperature for 1 hour. The membrane was incubated with Anti-Diphtheria Toxin antibody (abcam, lot NO: ab151222) diluted 2000 times in blocking buffer (4°C overnight), and the membrane was washed 3 times (5 minutes each time) with TBST solution (10mM Tris-HCl, 150mM NaCl, 0.1% Tween 20, pH7.4). The membrane was incubated with Goat Anti-Rabbit IgG H&L (HRP) antibody (abcam, lot NO: ab205718) diluted 10000 times in blocking buffer at room temperature for 1 hour. The membrane was washed three times with TBST (5 minutes each time) and then detected using chemiluminescence imaging (Bio-rad, ChemiDoc series imaging system).

[0120] The results are as follows Fig. 9 As shown. The results showed that both the CRM197 protein obtained by fermentation purification and the standard protein could bind to the rabbit polyclonal antibody of Diphtheria Toxin. Therefore, the antigenic epitope of the CRM197 protein obtained by fermentation purification was correctly retained and could be used for the production of polysaccharide conjugate vaccines.

[0121] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A nucleic acid construct comprising the following elements: Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, and sequence encoding CRM197 protein.

2. The nucleic acid construct according to claim 1, further comprising a gene encoding a selection marker; Preferably, the gene encoding the selection marker is located between the positive replication regulator gene and the E. coli DNA replication origin, or between the E. coli DNA replication origin and the nucleic acid sequence encoding the CRM197 protein; Preferably, the reading frame direction of the gene encoding the selection marker is opposite to the reading frame direction of the sequence encoding the CRM197 protein.

3. The nucleic acid construct according to any one of claims 1 to 2, further comprising a sequence encoding a signal peptide; Preferably, the sequence encoding the signal peptide is linked to the 5' end of the sequence encoding the CRM197 protein.

4. A nucleic acid construct according to any one of claims 1 to 3, comprising the following elements in sequence: Corynebacterium replicase gene, positive replication regulator gene, gene encoding selection marker, Escherichia coli DNA replication origin, sequence encoding signal peptide, and sequence encoding CRM197 protein; Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, gene encoding selection marker, sequence encoding signal peptide, and sequence encoding CRM197 protein; Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, antibiotic resistance gene, sequence encoding signal peptide, and DNA sequence encoding CRM197 protein; Corynebacterium replicase gene, positive replication regulator gene, antibiotic resistance gene, Escherichia coli DNA replication origin, sequence encoding signal peptide and DNA sequence encoding CRM197 protein; or Corynebacterium replicase gene, positive replication regulator gene, Escherichia coli DNA replication origin, kanamycin resistance gene, sequence encoding signal peptide and DNA sequence encoding CRM197 protein.

5. The nucleic acid construct according to any one of claims 1 to 4, characterized in that Any one or more of the following (1)-(6): (1) The Corynebacterium replicase gene is a Corynebacterium diphtheriae replicase gene; preferably, the Corynebacterium replicase gene encodes the amino acid sequence shown in SEQ ID NO:6; preferably, the Corynebacterium replicase gene comprises the sequence shown in SEQ ID NO:9 or SEQ ID NO:10; (2) the positive replication regulatory factor gene encodes the amino acid sequence shown in SEQ ID NO:7; preferably, the positive replication regulatory factor gene comprises the sequence shown in SEQ ID NO:11 or SEQ ID NO:12; (3) the E. coli DNA replication origin comprises the sequence shown in SEQ ID NO: 13; (4) The amino acid sequence of the CRM197 protein is shown in SEQ ID NO: 8; preferably, the DNA sequence encoding the CRM197 protein is shown in SEQ ID NO: 16 or SEQ ID NO: 3; (5) the gene encoding the selection marker is an antibiotic resistance gene; preferably, the antibiotic resistance gene is selected from one or more of an ampicillin resistance gene, a chloramphenicol resistance gene, a tetracycline resistance gene and a kanamycin resistance gene; and (6) The amino acid sequence of the signal peptide is shown in SEQ ID NO: 20; preferably, the sequence encoding the signal peptide is shown in SEQ ID NO:

21.

6. The nucleic acid construct according to any one of claims 1 to 5, comprising the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 in sequence; Preferably, the reading frame direction of the kanamycin resistance gene is opposite to the reading frame direction of the sequence encoding the CRM197 protein; Preferably, the sequence of the nucleic acid construct is as shown in SEQ ID NO:

16.

7. A vector comprising the nucleic acid construct according to any one of claims 1 to 6; preferably, the vector is a plasmid vector; preferably, the vector is a plasmid expression vector.

8. A recombinant host cell comprising the nucleic acid construct according to any one of claims 1 to 6 or the recombinant vector according to claim 7; preferably, the host cell is a recombinant Escherichia coli cell or a Corynebacterium cell such as a recombinant Corynebacterium diphtheriae cell; preferably, it is a recombinant Corynebacterium diphtheriae ATCC 39255 cell.

9. A cell culture comprising the recombinant host cell according to claim 12, or obtained by fermentation culture of the recombinant host cell according to claim 12.

10. A method for preparing CRM197 protein, comprising the steps of culturing the recombinant host cell according to claim 8 under suitable conditions, and isolating and purifying the CRM197 protein; preferably, isolating and purifying the CRM197 protein from the cell culture supernatant.

11. An isolated protein comprising the amino acid sequence shown in SEQ ID NO:

7.

12. An isolated nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:7; preferably, the isolated nucleic acid molecule comprises the sequence shown in SEQ ID NO:11 or SEQ ID NO:12.

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