Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori

A technology of genetically engineered vaccine and heat shock protein, which is applied in the field of purification process in the preparation of Helicobacter pylori heat shock protein A genetic engineering vaccine, and can solve the problems that the purification process cannot be directly applied

Inactive Publication Date: 2005-01-12
CHONGQING KANG WEI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to provide a simple process, high protein purity and good recovery rate for the existing purification process that cannot be directly applied to the recombinant Helicobacter pylori heat shock protein A genetic engineering vaccine protein constructed by the inventor. Purification process for preparation of recombinant Helicobacter pylori heat shock protein A genetic engineering vaccine

Method used

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  • Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori
  • Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori
  • Purification technique in preparing genetic engineering vaccine of heat shock protein A of recombined Helicobacter pylori

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Embodiment 1

[0031] The recombinant Helicobacter pylori heat shock protein A engineering bacterium constructed by the applicant was fermented at a high density, and the expression rate of the target protein was 23%. The bacteria were collected by centrifugation and purified according to the following steps:

[0032](1) Autoclaving: Suspend 500 g of highly expressed bacteria in TE nickel ion affinity chromatography buffer at a ratio of 1:10 (W / V), pre-cool at 4°C and mix with a cell homogenizer uniform. Use a high-pressure homogenizer to destroy bacteria under the condition of a pressure of 75Mpa (a total of 5 times, until the bacterial cracking rate is greater than 98%). After the bacteria are broken, take a small amount of bacterial liquid smear and stain, and observe the integrity of the cells under a microscope , to ensure that the cells are completely broken, centrifuge at 15,000g for 40min, collect the supernatant and discard the precipitate.

[0033] (2) Filtration: the above supern...

Embodiment 2

[0039] The sample treatment was the same as in Example 1, and the nickel ion affinity purification column Chelating Sepharose FF was used for preliminary purification, and the purification conditions were the same as in Example 1; 8mol / L urea and 50mmol / L DTT were used to denature and lyse the protein; under denaturing conditions, a gel filtration column Superdex 75 was used Purify (equilibrium buffer: 6mol / L guanidine hydrochloride, 20mmol Tris, pH8.5), and collect the target protein peak. Gel filtration column G-25 was used for denatured protein for desalting and refolding of denaturant, and refolding solution (20mmol NaHCO 3 / Na 2 CO 3 , 20mmol EDTA, pH10), and collect the target protein peak.

Embodiment 3

[0041] Sample treatment and purification conditions are the same as Example 1; denatured cleavage protein adopts 8mol / L urea and 50mmol / LDTT; under denaturing conditions, adopt gel filtration column Superdex 200 to purify (equilibrium buffer: 6mol / L guanidine hydrochloride, 20mmol Tris, pH8. 5), collect the target protein peak; HspA denatured protein, use the nickel ion affinity chromatography column Chelating Sepharose HP to carry out on-column renaturation, the method is that the chromatography column is equilibrated with equilibrium solution (8mol / L urea, 20mmol Tris, pH 8.5) Afterwards, load the denatured target protein sample, use refolding solution (20mmol / L PBS, 2mmol GSH, 0.2mmol / L GSSG, 0.15mol / L NaCl) to slowly gradient elute the column until urea is completely removed, use imidazole buffer (20mmol / L PBS, 0.5mol / L imidazole, pH8.5) to elute the target protein and collect the protein peak.

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Abstract

This invention disclose a purification technology in the preparation of reconstitution pylorus spirillum heat shock protein A gene engineering vaccines applying pressure, filtration, Ni ions affinity chromatography and purification, concentration lysis, glue filtration chromatography and renaturation to get pure reconstitution pylorus spirillus heat shock protein A.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a purification process in the preparation of Helicobacter pylori heat shock protein A genetic engineering vaccine. technical background [0002] Helicobacter pylori (Hp) is a Gram-negative, screw-shaped, microaerophilic bacterium that colonizes the stomach. The infection rate in the world population exceeds 50%. , peptic ulcer and non-ulcer dyspepsia are the main pathogenic factors, and are closely related to the occurrence of gastric cancer. [0003] Known Hp strains can produce two heat shock proteins, HspA and HspB. HspA is composed of 118 amino acids, 91 amino acids at the N-terminus have high homology with E. coli GroES, and can be used as molecular chaperones, playing an important role in the synthesis and assembly of Hp protein; the 27 amino acid regions at the C-terminus are unique to Hp and rich in Contains histidine and cysteine, is a nickel ion binding domain and is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/36C07K14/195C12P21/00
Inventor 邹全明郭鹰冉向阳朱永红吴超张卫军童文德
Owner CHONGQING KANG WEI BIOTECH
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