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Diphtheria toxin mutant with characteristic of soluble high expression in Escherichia coli

A technology of diphtheria toxin and Escherichia coli, which is applied in the field of genetic engineering, can solve the problems of high fermentation cost, harsh fermentation conditions of diphtheria bacteria, and low soluble expression, and achieve the effect of broad application prospects

Active Publication Date: 2017-01-25
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing preparation method of CRM197 mainly contains three kinds: (1) form lysogenized bacterial strain with the mutant β bacteriophage lysogenous diphtheriae bacillus, secrete CRM197, its shortcoming is that yield is low, diphtheriae bacillus fermentation condition is harsh, fermentation cost is higher, and Medium components are complex and expensive
(2) Construct a recombinant plasmid containing the CRM197 base mutation sequence and introduce it into diphtheria bacteria for expression. The disadvantage is that the yield has not been greatly improved, and the fermentation cost is still high
The current problem is that the soluble expression is low, which is not enough to meet the needs of industrial production
Invention patents CN201310219018.6, CN201310163343.5, CN101265288B and CN100519577C all disclosed the expression and purification of CRM197 in Escherichia coli, but they are all expressed in inclusion bodies, which require denaturation and renaturation, and cannot directly obtain CRM197 with a natural conformation

Method used

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  • Diphtheria toxin mutant with characteristic of soluble high expression in Escherichia coli
  • Diphtheria toxin mutant with characteristic of soluble high expression in Escherichia coli
  • Diphtheria toxin mutant with characteristic of soluble high expression in Escherichia coli

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of diphtheria toxin mutant CRM197

[0032] 1. Sequence optimization of CRM197

[0033] According to the diphtheria toxin sequence published by GenBank No.K01722.1, the CRM197 sequence of 535Aa was analyzed and optimized, the common codons of Escherichia coli were used to replace the rare codons, and the ratio and distribution of the four bases A, T, G, and C were balanced. EcoRI and XhoI restriction sites were added at both ends of the CRM197 sequence, and a TAA stop codon was added before the start codon to terminate the expression of Trx protein on the vector pET32a(+). The optimized sequence is shown in SEQ ID NO.1, and the nucleotide sequence comparison before and after optimization is shown in Figure 1A and Figure 1B , in the optimized row, the underlined bases are optimized bases. The optimized nucleotide sequence is subjected to whole gene synthesis.

[0034] 2. Construction of CRM197 expression vector

[0035]The optimized and synthe...

Embodiment 2

[0043] Embodiment 2: Toxicity test of recombinant CRM197 protein (Vero cell method)

[0044] After the Vero cells were digested, the cells were counted, and the cell concentration was adjusted to 1.25×10 5 cells / mL, the cell suspension was added to a 96-well culture plate, 100 μL / well. Diphtheria toxoid was serially diluted from 10ng / mL, and added to the culture plate containing cells sequentially, 100μL / well; diphtheria toxoid and CRM197 protein were serially diluted from 0.5mg / mL, and added to the culture plate containing cells sequentially , 100 μL / well. The Vero cell culture plate added with toxin, toxoid and CRM197 protein was further cultured in a carbon dioxide incubator at 37°C for 6-7 days, and the survival of the cells was observed by the MTT method. From Figure 5 The experimental results can be seen, compared with diphtheria toxin, CRM197 dose increased 5 × 10 7 There was no toxicity after 10 times; compared with diphtheria toxoid, the dose of CRM197 was increa...

Embodiment 3

[0045] Embodiment 3, the immunogenicity research of CRM197

[0046] Different doses of CRM197 protein 2μg and 20μg / mouse were used to immunize 6-8 week-old Balb / c mice, once every two weeks, for a total of three times, all with aluminum hydroxide adjuvant, two weeks after each immunization, and the next time Blood was taken before immunization to detect the antibody titer in the serum; the antibody titer was determined by ELISA method, diphtheria toxoid 2 μg / mL, 100 μL / well coated with 96-well enzyme-linked plate, and coated overnight at 4 °C. Wash with PBST 4 times, 5min / time. The antiserum was diluted sequentially from 1:100 with PBST, added to the enzyme-linked plate, and reacted at 37°C for 1 hour, and the serum immunized with PBS was used as the control. Wash with PBST 4 times, 5min / time. Add HRP-anti-mouse secondary antibody and react at 37°C for 30-40min. Add TMB color development solution, 50 μL / well, after color development, use 2M H 2 SO 4 Termination, microplat...

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Abstract

The invention discloses a dendritic toxin mutant CRM197 coding sequence, the nucleotide sequence of which is optimized. The synthesized coding sequence is ligated into an expression vector pET32a(+) after double restriction endonuclease digestion, and soluble highly expressed recombinant CRM197 is obtained in Escherichia coli BL21(DE3). Compared with diphtheria toxin, the recombinant CRM197 protein prepared according to the invention is safe and non-toxic and can induce high titer protective antibody in mice, and has broad application prospect in large-scale and efficient preparation of recombinant CRM197.

Description

technical field [0001] The invention discloses a soluble highly expressed diphtheria toxin mutant and its optimized nucleotide coding sequence, belonging to the technical field of genetic engineering. Background technique [0002] Diphtheria toxin (Diphtheria Toxin, DT) contains 535 amino acids and has a molecular weight of about 58KD. It is an exotoxin produced by Corynebacterium diphtheria infected with the genome of β-bacteriophage. The toxin can be hydrolyzed by trypsin to form a fragment A of 193 amino acid residues and a fragment B of 342 amino acid residues. [0003] CRM197 is a mutant of diphtheria toxin. CRM197 has a complete diphtheria toxin functional structure, but because its 52nd amino acid is mutated from Gly to Glu, it loses diphtheria toxin enzyme activity and toxicity, but still retains the immunogen of diphtheria toxin sex. CRM197 can be used in many fields: (1) In the process of polysaccharide vaccine preparation, CRM197 is often used as an immune prote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N1/21C07K14/34C12R1/19
CPCC07K14/34C12N15/70C12N2800/101
Inventor 陈薇于蕊徐俊杰候利华于长明李建民付玲房婷张金龙宋小红刘树玲杨秀旭
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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