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Group B Neisseria meningitidis recombinant pilin Fim, and preparation method and application thereof

A Neisseria and meningitis technology, applied in the field of Neisseria meningitidis recombinant pili protein Fim and its preparation, can solve the problems of vaccine research and marketing, and achieve good repeatability, high recovery rate, good The effect of the bactericidal effect

Active Publication Date: 2020-06-19
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant vaccine research on the prevention of meningitis by blocking the adhesion of Neisseria meningitidis pili

Method used

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  • Group B Neisseria meningitidis recombinant pilin Fim, and preparation method and application thereof
  • Group B Neisseria meningitidis recombinant pilin Fim, and preparation method and application thereof
  • Group B Neisseria meningitidis recombinant pilin Fim, and preparation method and application thereof

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preparation example Construction

[0053] Further, the present invention also provides a method for preparing the above-mentioned Neisseria meningitidis recombinant pilus protein Fim, comprising the following steps:

[0054] a. Plasmid construction

[0055] The gene whose nucleotide sequence is SEQ ID NO:1 is connected in the expression vector plasmid, the plasmid is constructed and transferred into the host bacteria for induced expression;

[0056] b. Sonication, centrifugation

[0057] The bacteria obtained in the collection step a were resuspended and mixed evenly with the bacteriostasis solution, and after an ice-water bath, the bacteria were destructed by ultrasonication, centrifuged at a high speed, and the supernatant was collected;

[0058] c. Ni column affinity purification

[0059] Preliminary purification with Ni affinity packing, use liquid A to equilibrate the chromatographic column, and use liquid B to elute;

[0060] d. SP column affinity chromatography;

[0061] The target protein purified i...

Embodiment 1

[0080] Construction and identification of the recombinant plasmid pET28a / Fim of embodiment 1 Fim gene

[0081] The specific operation steps are as follows:

[0082] (1) According to the complete genome sequence of Neisseria meningitidis group B, the candidate antigen Fim (SEQ ID NO: 2) with protective immune response was screened by reverse vaccinology.

[0083] (2) According to the amino acid sequence of Fim, Escherichia coli partial tropism codon optimization was performed to obtain the target gene fragment, the sequence is SEQ ID NO: 3 (the base sequence of the restriction site is underlined).

[0084] Nucleotide sequence of SEQ ID NO:3 target gene

[0085] ccatggcggaaggccagaaaagcgcggtgaccgaatattatctgaaccacggagaatggccgggcaacaacagcagcgcgggcgtggcgaccagcgcggatattaaaggcaaatatgtgaaaagcgtggaagtgaaaaacggcgtggtgaccgcgcagatggcgagcagcaacgtgaacaacgaaattaaaggcaaaaaactgagcctgtgggcgaaacgtcaggcgggcagcgtgaaatggttttgcggcctgccggtgacccgtgcggataacgcgaaagatgatgcggtgaccgcggcggcgaccggcaccgataa...

Embodiment 2

[0097] Example 2 Induced expression, purification and identification of expression form of recombinant pilus protein Fim in prokaryotic expression system-Escherichia coli The specific operation steps are as follows:

[0098] (1) Take 100 μl of overnight cultured pET28a / Fim / BL21(DE3) bacterial solution and add it to 10 mL of kanamycin + resistant LB medium, culture overnight at 220 rpm at 37°C, take 200 μl of overnight cultured bacterial solution and add 20 mL card In the LB medium with Namycin + resistance, culture at 220rpm 37℃ for 2h, after secondary activation to OD600 of 0.8, add IPTG Isopropyl-β-D-thiosemi lactoside 10 μl to make the final concentration 0.5 mM, and then placed on a shaker at 220 rpm at 37° C. to induce expression for 4 hours.

[0099] (2) Take out the bacterial solution after induced expression, centrifuge at 5000g for 15min, discard the supernatant, add 3ml of bacteriostasis solution (50mM PB, 0.3M NaCl, pH7.4) and mix evenly, ultrasonically lyse in i...

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Abstract

The invention belongs to the technical field of biopharmaceutics, and in particular to a group B Neisseria meningitidis recombinant pilin Fim, and a preparation method and application thereof. The present market lacks a vaccine which directly blocks adhesion of Neisseria meningitidis pilin to prevent meningitis. The invention provides a group B Neisseria meningitidis recombinant pilin Fim gene with a nucleotide sequence shown as SEQID NO:1. The group B Neisseria meningitidis recombinant pilin Fim gene is first found to be a key molecule for adhesion and colonization of Neisseria meningitidis.The group B Neisseria meningitidis recombinant pilin Fim gene is cloned into a protein expression vector and then is expressed on a large scale, and the recombinant protein is prepared after purification, wherein the purity of the recombinant protein is high and reaches 90% or above. The repeatability is good and the recovery rate is high. The protein can be used as a vaccine candidate to preventinfection of group B Neisseria meningitides, has the advantages of soluble expression, easy purification, high purity and simple preparation method, and has notable economic benefits.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a recombinant pili protein Fim of Neisseria meningitidis group B and its preparation method and application. Background technique [0002] Meningococcus, scientific name Neisseria meningitidis (n.meninyitidis), is the pathogenic bacteria of epidemic cerebrospinal meningitis (meningitis). It is a Gram-negative diplococcus that exists in the cerebrospinal fluid of patients with acute or early meningitis, mostly located in neutrophils, and is kidney-shaped, arranged in pairs, with opposite concave surfaces. Virulent strains of meningococci can be observed under the electron microscope to have microcapsules and pili. [0003] Humans are the only susceptible host for N. meningitidis. Bacteria invade the body from the nasopharynx, relying on the action of pili to adhere to the surface of the epithelial cells of the nasopharyngeal mucosa. The pathogenesis of Ne...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C07K14/22C07K1/22A61K39/095A61P31/04
CPCA61K39/095A61P31/04C07K14/22C12N15/70
Inventor 刘开云刘宇郭刚李彦王宁石云舒月力
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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