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CN-121991191-A - Soluble recombinant diphtheria toxin mutant CRM197 protein and preparation method thereof

CN121991191ACN 121991191 ACN121991191 ACN 121991191ACN-121991191-A

Abstract

The invention belongs to the field of genetic engineering, and particularly discloses a soluble recombinant diphtheria toxin mutant CRM197 protein and a preparation method thereof, wherein the specific steps include connecting a codon-optimized diphtheria toxin mutant CRM197 coding sequence into an expression vector pBAD-HisB and inducing expression in escherichia coli BW25113, converting the diphtheria toxin mutant CRM197 from inclusion bodies into soluble expression by adding a signal peptide sequence at the N end, and obtaining the protein purity reaching more than 95% and the yield reaching more than 600 mg/L after fermentation optimization and multi-step purification.

Inventors

  • HAN JIAN
  • ZHONG DEYI
  • LIU CONG
  • HAN WENQING
  • HU KAILEI
  • XU ZHIKUN
  • WEI HAITAO

Assignees

  • 杭州中科奥美生物科技有限公司

Dates

Publication Date
20260508
Application Date
20260120

Claims (7)

  1. 1. A method for preparing soluble recombinant diphtheria toxin mutant CRM197 protein, comprising the steps of: S1, optimizing the sequence of the diphtheria toxin mutant CRM197 by using escherichia coli preference codons, wherein the amino acid sequence of the diphtheria toxin mutant CRM197 after the optimization of the escherichia coli preference codons is shown as SEQ ID NO. 1; s2, constructing an expression vector containing an optimized diphtheria toxin mutant CRM197, and connecting a signal peptide PelB or OmpF at the 5' end of the sequence, wherein the amino acid sequence of the signal peptide PelB is shown as SEQ ID NO. 2, and the amino acid sequence of the signal peptide OmpF is shown as SEQ ID NO. 3; S3, converting the expression vector constructed in the step S2 into an escherichia coli host cell to obtain engineering bacteria, culturing the engineering bacteria and inducing to express a soluble recombinant diphtheria toxin mutant CRM197 protein; s4, crushing thalli, and collecting supernatant containing soluble recombinant diphtheria toxin mutant CRM197 protein; S5, purifying the supernatant to obtain the high-purity soluble recombinant diphtheria toxin mutant CRM197 protein.
  2. 2. The method for preparing soluble recombinant diphtheria toxin mutant CRM197 protein according to claim 1, wherein the expression vector in step S2 is pBAD-HisB.
  3. 3. The method of claim 1, wherein the supernatant of step S5 is purified by anion exchange chromatography and heparin affinity chromatography.
  4. 4. A soluble recombinant diphtheria toxin mutant CRM197 protein produced by the production process of any one of claims 1-3.
  5. 5. A group B meningococcal fHBP fusion protein is characterized in that the soluble diphtheria toxin mutant CRM197 protein and a group B meningococcal fHBP variant fragment are connected through a flexible peptide, wherein the group B meningococcal fHBP variant fragment is fHBP-V1 or fHBP-V2, the amino acid sequence of the fHBP-V1 is shown as SEQ ID NO. 4, and the amino acid sequence of the fHBP-V2 is shown as SEQ ID NO. 5.
  6. 6. The group B meningococcal fHBP fusion protein of claim 5, wherein the flexible peptide has an amino acid sequence of 5-40 serine and glycine in random combination.
  7. 7. The meningococcal fHBP fusion protein of claim 5, wherein the soluble diphtheria toxin mutant CRM197 protein is C-terminally linked to the N-terminus of fHBP-V1 or fHBP-V2, or the C-terminus of fHBP-V1 or fHBP-V2 is C-terminally linked to the N-terminus of the soluble diphtheria toxin mutant CRM197 protein.

Description

Soluble recombinant diphtheria toxin mutant CRM197 protein and preparation method thereof Technical Field The invention belongs to the field of genetic engineering, and particularly relates to a soluble recombinant diphtheria toxin mutant CRM197 protein and a preparation method thereof. Background CRM197 (Cross-REACTING MATERIALS 197) is a mutant of diphtheria toxin, has a complete diphtheria toxin functional structure, is composed of two subunits (connected by disulfide bonds), has almost the same overall folding of CRM197 and diphtheria toxin, but has no enzyme activity and toxicity of diphtheria toxin, does not generate diphtheria toxoid toxicity reversal effect due to the fact that the 52 th amino acid is mutated from Gly to Glu, has immunogenicity of diphtheria toxin, and retains a potential binding site of a complete lysine amino structure, so that the CRM197 and diphtheria toxin are often used as an ideal immune protein carrier and widely applied to the field of vaccine development. The existing preparation method of diphtheria toxin mutant CRM197 mainly comprises three steps of 1) secretion of CRM197 by using mutant diphtheria bacillus, and has the defects of low yield, higher cost of fermentation equipment, complex culture medium components and high price. 2) The recombinant plasmid containing diphtheria toxin mutant CRM197 base mutation sequence is constructed to be introduced into diphtheria bacillus for expression, and the disadvantage is that the yield is not greatly improved yet and the fermentation cost is still higher. 3) Constructing recombinant plasmid containing diphtheria toxin mutant CRM197 base mutation sequence, and introducing the recombinant plasmid into escherichia coli for prokaryotic expression. The expression of the diphtheria toxin mutant CRM197 by using escherichia coli has important significance, and has the characteristics of high yield, simplicity, rapid growth, expression of an oxidation host, simultaneous expression of chaperones, low endotoxin level, periplasm output of cells and the like. The existing problem is that in an escherichia coli expression system, the recombinant diphtheria toxin mutant CRM197 is mainly expressed in an inclusion body form, and the soluble expression is low, so that the requirements of industrial production and practical application are not met. Neisseria meningitidis group B (NEISSERIA MENINGITIDIS B, men B) is a gram-negative coccus, and because of its capsular polysaccharide and human neural cell adhesion molecular structure are similar, the immunogenicity is poor, and effective prevention cannot be performed by means of conventional polysaccharide vaccines, and its vaccine development mainly depends on outer membrane protein antigens. Wherein, the H factor binding protein (fHBP) is a key vaccine candidate antigen, and can be divided into three variants of V1, V2 and V3 according to sequence differences, and immunodominant epitopes of the H factor binding protein (fHBP) are respectively positioned in different structural domains. Currently, vaccines (e.g., trumenba, BEXSERO) and chinese patent No. CN106659776a attempt to construct fusion proteins using fHBP variants, but still suffer from insufficient immunogenicity or limited strain coverage. Therefore, a new strategy is needed to efficiently prepare soluble CRM197 and fuse it with fHBP antigen to enhance vaccine immunity. Disclosure of Invention In order to solve the problems in the prior art, the invention provides a soluble recombinant diphtheria toxin mutant CRM197 protein and a preparation method thereof. The technical scheme of the invention is as follows: the invention aims at providing a preparation method of soluble recombinant diphtheria toxin mutant CRM197 protein, which comprises the following steps: S1, optimizing the sequence of the diphtheria toxin mutant CRM197 by using escherichia coli preference codons, wherein the nucleotide sequence of the diphtheria toxin mutant CRM197 after the optimization of the escherichia coli preference codons is shown as SEQ ID NO. 1; s2, constructing an expression vector containing an optimized diphtheria toxin mutant CRMA197, and connecting a signal peptide PelB or OmpF at the 5' end of the sequence, wherein the amino acid sequence of the signal peptide PelB is shown as SEQ ID NO. 2, and the amino acid sequence of the signal peptide OmpF is shown as SEQ ID NO. 3; s3, converting the expression vector constructed in the step S2 into an escherichia coli host cell to obtain engineering bacteria, culturing the engineering bacteria and inducing to express soluble recombinant diphtheria toxin mutant CRM197 protein; s4, crushing thalli, and collecting supernatant containing soluble recombinant diphtheria toxin mutant CRM197 protein; S5, purifying the supernatant to obtain the high-purity soluble recombinant diphtheria toxin mutant CRM197. Preferably, the expression vector described in step S2 is pBAD-HisB. Preferably, the supernatant in step S5 is pur