CN104592366A - Preliminary purification method for chicken infectious bursa disease virus VP2 protein - Google Patents
Preliminary purification method for chicken infectious bursa disease virus VP2 protein Download PDFInfo
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- CN104592366A CN104592366A CN201410812737.3A CN201410812737A CN104592366A CN 104592366 A CN104592366 A CN 104592366A CN 201410812737 A CN201410812737 A CN 201410812737A CN 104592366 A CN104592366 A CN 104592366A
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- ammonium sulfate
- purification method
- preliminary purification
- supernatant
- protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
- C07K14/08—RNA viruses
Abstract
The invention discloses a preliminary purification method for a chicken infectious bursa disease virus VP2 protein. The method comprises the following steps: (1) treating thalli; (2) adding ammonium sulfate into a supernatant to 20% saturation degree, stirring on ice till ammonium sulfate is fully dissolved and standing overnight, centrifugalizing for 15 minutes at 8000rpm and 4 DEG C and collecting the supernatant; (3) adding ammonium sulfate into the supernatant in the step (2) to 50% saturation degree, stirring on ice till ammonium sulfate is fully dissolved and standing overnight, centrifugalizing for 15 minutes at 8000rpm and 4 DEG C and collecting a precipitate; and (4) drying in vacuum to obtain the chicken infectious bursa disease virus VP2 protein. The invention provides the method which is simple to operate and low in cost.
Description
Technical field
The present invention relates to purified technology of protein field, be specifically related to a kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method.
Background technology
Along with the development of Protocols in Molecular Biology, recombinant protein biological products are exploited gradually.Infections chicken cloacal bursa virus VP2 albumen is based on Escherichia coli system expression, but foreign gene is the inevitable expression with host and other gene in expression process, so need infections chicken cloacal bursa virus VP2 albumen to be purified from multiple protein plastome, and traditional purification process is to after thalline process, supernatant is splined on nickel post, post bed is repeatedly rinsed 3 times with the lavation buffer solution of same column bed volume, use the elution buffer wash-out of same column volume again, collect eluting liquid and put into dialysis tubing, dialyzed overnight, the recombination chicken Infectious bursal disease virus VP2 dry powder of acquisition is after vacuum-drying, the organic efficiency of the method is low, and method complexity is not easy to operate, and cost is higher.
Summary of the invention
The object of the invention is the shortcoming and defect in order to overcome prior art, for setting up a kind of purifying process simple to operate, with low cost, providing a kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method.
A kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method, comprises the following steps:
(1) thalline process;
(2) in supernatant, add ammonium sulfate to 20% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 15min of the rotating speed of 8000rpm at 4 DEG C of temperature, collects supernatant;
(3) in the supernatant of step 2, add ammonium sulfate to 50% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 15min of the rotating speed of 8000rpm at 4 DEG C of temperature, collecting precipitation;
(4) vacuum-drying, is the recombination chicken Infectious bursal disease virus VP2 of acquisition afterwards.
The invention has the beneficial effects as follows: after the method process, often liter of zymocyte liquid finally can obtain recombination chicken Infectious bursal disease virus VP2 3.8g, compared with existing affinity chromatography technology, have simple to operate, cost is low, efficiency advantages of higher.For large-scale production lays the foundation.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail:
One, test materials and instrument:
Infections chicken cloacal bursa virus positive serum, infections chicken cloacal bursa virus standard agp antigen detect institute purchased from Chinese veterinary medicament, whizzer (3K15) available from Sigma, magnetic stirring apparatus is purchased from the new sesame in Ningbo, 5L intelligence fermentation reactor converges hall purchased from Shanghai, and protein purification test kit is purchased from German NOVAGEN;
Lavation buffer solution: phosphoric acid salt 20 mM, NaCl0.5 M, imidazoles 20-40 mM, pH 7.4;
Elution buffer: phosphoric acid salt 20 mM, NaCl0.5 M, imidazoles 500 mM, pH 7.4.
Two, experimental technique:
(1) thalline process: picking e. coli bl21/pET28a-VP2 bacterium colony, be inoculated in 5mlLB(containing kana, working concentration is 50 μ g/ml) in test tube, 37 DEG C of shaking culture are spent the night.Next day is inoculated into 100mlLB(containing kana according to the ratio of 1:100, working concentration is 50 μ g/ml) in substratum 37 DEG C cultivate 16-18h, then be seeded in 5L fermentor tank (containing kana, working concentration is 50 μ g/ml), when to be cultured to OD value be 0.8, adding lactose to final concentration is 40mmol/L inducing culture 5h.Centrifugal 10min under cultivation bacterium liquid is placed at 5000rpm rotating speed, collects thalline 50g.Add the calculating of 10ml lysate by every gram of thalline and add lysate 500ml altogether, add N,O-Diacetylmuramidase (working concentration is 100ug/mL) and be placed on 37 DEG C of water-bath 1h, multigelation three times.After ice-bath ultrasonic fragmentation, 8000rpm centrifuging and taking supernatant;
(2) in supernatant, add ammonium sulfate to 20% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 15min of the rotating speed of 8000rpm at 4 DEG C of temperature, collects supernatant;
(3) in the supernatant of step 2, add ammonium sulfate to 50% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 15min of the rotating speed of 8000rpm at 4 DEG C of temperature, collecting precipitation;
(4) vacuum-drying, is the recombination chicken Infectious bursal disease virus VP2 of acquisition afterwards.
Three, experimental result contrast:
Redissolved with 500mL physiological saline by the recombination chicken Infectious bursal disease virus VP2 lyophilized powder of experiment gained, carry out agar immunodiffusion test after redissolution, compared the organic efficiency of two kinds of methods by antigen valence, result is as following table:
| Solution after infections chicken cloacal bursa virus VP2 protein freeze-dried powder redissolves | Ammonium sulfate precipitation method preparation of the present invention | Conventional nickel column method preparation |
| Result | 2 7 | 2 4 |
Claims (4)
1. an infections chicken cloacal bursa virus VP2 albumen preliminary purification method, is characterized in that, comprise the following steps:
(1) thalline process;
(2) in supernatant, add ammonium sulfate to 10%-30% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 10-20min of the rotating speed of 8000rpm at 4 DEG C of temperature, collects supernatant;
(3) in the supernatant of step (2), add ammonium sulfate to 40%-60% saturation ratio, be stirred to after ammonium sulfate dissolves hold over night completely on ice, the centrifugal 10-20min of the rotating speed of 8000rpm at 4 DEG C of temperature, collecting precipitation;
(4) vacuum-drying, is the recombination chicken Infectious bursal disease virus VP2 of acquisition afterwards.
2. a kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method according to claim 1, is characterized in that, ammonium sulfate to 20% saturation ratio that described step (2) is added.
3. a kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method according to claim 1, is characterized in that, ammonium sulfate to 50% saturation ratio that described step (3) is added.
4. a kind of infections chicken cloacal bursa virus VP2 albumen preliminary purification method according to claim 1, it is characterized in that, described step (2) and the centrifugation time of step (3) are 15min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410812737.3A CN104592366A (en) | 2014-12-24 | 2014-12-24 | Preliminary purification method for chicken infectious bursa disease virus VP2 protein |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410812737.3A CN104592366A (en) | 2014-12-24 | 2014-12-24 | Preliminary purification method for chicken infectious bursa disease virus VP2 protein |
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| CN104592366A true CN104592366A (en) | 2015-05-06 |
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| CN201410812737.3A Pending CN104592366A (en) | 2014-12-24 | 2014-12-24 | Preliminary purification method for chicken infectious bursa disease virus VP2 protein |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138517A (en) * | 2020-01-10 | 2020-05-12 | 武汉科前生物股份有限公司 | Method for improving safety of recombinant protein vaccine and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688487A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease virus (IBD) and viral arthritis (VA) four-joint inactivated vaccine |
| CN102994520A (en) * | 2012-12-03 | 2013-03-27 | 青岛康地恩药业股份有限公司 | Infectious bursal disease VP2 gene and application thereof |
| CN103667199A (en) * | 2012-09-20 | 2014-03-26 | 厦门大学 | Method for preparing rotavirus bilayer virus-like particles in vitro |
-
2014
- 2014-12-24 CN CN201410812737.3A patent/CN104592366A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102688487A (en) * | 2012-06-18 | 2012-09-26 | 青岛易邦生物工程有限公司 | Production method of newcastle disease (ND), infectious bronchitis (IB), infectious bursal disease virus (IBD) and viral arthritis (VA) four-joint inactivated vaccine |
| CN103667199A (en) * | 2012-09-20 | 2014-03-26 | 厦门大学 | Method for preparing rotavirus bilayer virus-like particles in vitro |
| CN102994520A (en) * | 2012-12-03 | 2013-03-27 | 青岛康地恩药业股份有限公司 | Infectious bursal disease VP2 gene and application thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138517A (en) * | 2020-01-10 | 2020-05-12 | 武汉科前生物股份有限公司 | Method for improving safety of recombinant protein vaccine and application thereof |
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Application publication date: 20150506 |