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Purification method for 69KD outer membrane protein of pertussis bacillus

A technology for outer membrane protein and whooping cough, applied in the field of protein separation and purification, can solve the problems of high cost of materials, many steps, and long time-consuming dialysis steps, and achieve the effect of low cost and high-efficiency inoculation

Inactive Publication Date: 2012-07-18
北京天坛生物制品股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows scientists to create an easy way to make proteins that are very similar but different from other molecules found naturally on Earth. It makes it easier than traditional ways like chemical extraction or crystallization techniques because this technique does away with complicated steps involved beforehand.

Problems solved by technology

Technologies described in these technical problem addressed in this patents involve developing efficient techniques for producing pure specific forms of target molecules called precursors of Portebronchuetzler Neisseriamiditis Viruses (PNR). Current technological processes require multiple steps involving several hours, making mass product manufacture difficult and expensive.

Method used

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  • Purification method for 69KD outer membrane protein of pertussis bacillus
  • Purification method for 69KD outer membrane protein of pertussis bacillus
  • Purification method for 69KD outer membrane protein of pertussis bacillus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1. the selection of thalline treatment method and the optimization of condition

[0068] 1.1 Acquisition of bacteria:

[0069] Cultivate the CS bacterial strain (CMCC58003, purchased from ATCC (American Type Microorganism Collection Center)) in 10L S-S medium to a cell concentration of 22 billion / ml, centrifuge at 8000rpm for 10 minutes to obtain the cell, and use 0.035mol / L Sodium chloride, 1mmol / L ethylenediaminetetraacetic acid (EDTA), 0.025mol / L Tris-HCl buffer solution of pH 8.8 of 1mmol / L phenylmethylsulfonyl fluoride (PMSF) dissolves bacterium, obtains 0.05g / ml of bacterial suspension, and processed according to the following methods.

[0070] 1.2 Three ways to deal with bacteria:

[0071] A. Concentrated salt solution leaching supernatant: Suspend the bacteria in Tris-HCl buffer solution containing 0.5mol / L NaCl, put in a water bath at 60°C for 1 hour, centrifuge at 8500r / min at 4°C for 10 minutes, and take the supernatant.

[0072] B. Obtain the ...

Embodiment 2

[0086] Embodiment 2. The selection of crude extraction method and the optimization of conditions

[0087] 2.1 Ethanol precipitation crude extraction method:

[0088] Through the above optimized conditions (1mol / LNaCl, incubate at 60°C for 1 hour) to obtain concentrated salt solution extract, pre-cool 2 times the volume of ethanol at -20°C for 30 minutes, then slowly add ethanol to the extract, and stir evenly , -20°C for 30 minutes. 10000r / min, 4°C, centrifuged for 30 minutes, and the precipitate obtained by centrifugation was mixed with 0.035mol / L sodium chloride, 1mmol / L ethylenediaminetetraacetic acid (EDTA), 1mmol / L phenylmethylsulfonyl fluoride (PMSF) 0.025 mol / L Tris-HCl buffer solution of pH 8.8 was dissolved, and dialyzed against this buffer solution overnight.

[0089] 2.2 Ammonium sulfate precipitation:

[0090] Add ammonium sulfate to the concentrated salt solution extract obtained by the above method (1mol / LNaCl, incubate at 60°C for 1 hour) to make the concentr...

Embodiment 3

[0095] Example 3: Determination of the column chromatography purification method of fine extraction and optimization of conditions

[0096] 3.1 Affinity gum blue (AGB) column chromatography:

[0097] Under the above-mentioned conditions, leaching with concentrated salt and passing through 20% by weight / volume and 33% by weight / volume of ammonium sulfate fractional precipitation obtains the precipitate and dissolves, and the obtained protein crude extract is in 20mmol / L Tris-HCl (pH7.4 ) buffer, stirred at 4°C, and equilibrated. Wash the chromatographic column with an aqueous solution of potassium dihydrogen phosphate with a concentration of 0.5 mmol / L at pH 7.5 to remove impurity proteins, and use pH 8.0 containing 4 mol / L urea, 0.15 mol / L sodium chloride, 1 mmol / LEDTA, 1 mmol / L L PMSF and 10mmol / L Tris-HCl buffer to elute the target protein.

[0098] The results after electrophoresis of the samples collected after the AGB column are as follows: Image 6 As shown, lanes A t...

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Abstract

The invention belongs to the field of protein separating purification and relates to an industrialized large-scale separating purification method of 69KD outer membrane protein of pertussis bacillus. The method comprises extracting pertussis bacillus CS thallus in buffer solution with potential of hydrogen (pH) 8.8 and containing sodium chloride of 1mol/L to 2mol/l for thirty minutes to three hours at 40 to 60 DEG C, performing centrifugation, collecting supernatant, and obtaining thallus lysate supernatant of the 69KD outer membrane protein; b adding ammonium sulfate into the thallus lysate supernatant of the 69KD outer membrane protein to enable the ammonium sulfate to achieve 15% weight/volume to 25% weight/volume, standing the solution for one hour to eight hours, performing centrifugation, and collecting the supernatant; c adding ammonium sulfate into the supernatant collected in step b to enable the ammonium sulfate to achieve 30% weight/volume to 38% weight/volume, standing the solution for one hour to eight hours, performing centrifugation, abandoning the supernatant, and collecting deposit; d dissolving the deposit collected in the step c through Tris-HCl buffer solution, and dialyzing the solution to remove the ammonium sulfate; and e enabling the solution to pass through a diethyl aminoethyl (DEAE) sepharose ion exchange column, utilizing buffer solution with linear gradient sodium chloride concentration from 0 mol/L to 1 mol/L for washing, and collecting objective protein. The purification method for the 69KD outer membrane protein of the pertussis bacillus can fast and conveniently purify natural conformation 69 KD outer membrane protein so that the method can be applied to large-scale industrial preparation of the natural configuration 69 KD outer membrane protein.

Description

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Claims

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Application Information

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Owner 北京天坛生物制品股份有限公司
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