Method for efficiently expressing avian leukosis P27 protein and application thereof

A high-efficiency expression technology for avian leukemia, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of unsatisfactory kit production and quality inspection, unstable expression and yield, time-consuming and laborious Product and other issues, to achieve the effect of efficient expression, shortened production cycle, and long preparation time

Pending Publication Date: 2022-05-10
哈尔滨国生生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the expression of avian leukemia P27 protein. The expression systems used include Escherichia coli and baculovirus expression systems, but they are all expressed in small quantities. The unit volume is generally 300ml-1000ml, and the expression amount is about 40mg / L; the expression concentration is low. ; After purification, the yield should only reach 80 mg / batch; and there are large batch-to-batch differences, and the expression and yield are not stable each time. Not only is it time-consuming and laborious, but the product cannot meet the requirements of kit production and quality inspection. need

Method used

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  • Method for efficiently expressing avian leukosis P27 protein and application thereof
  • Method for efficiently expressing avian leukosis P27 protein and application thereof
  • Method for efficiently expressing avian leukosis P27 protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Induction of Recombinant Escherichia coli BL21(DE3)-pET-30a-p27 by Shaker Culture

[0105] Strain culture: Take a BL21(DE3)-pET-30a-p27 glycerol strain stored at -80°C, use an inoculation loop to dip the strain into four sections on the LB plate, and culture overnight at 37°C;

[0106] Preparation of first-level seed liquid: Take the monoclonal cultured overnight in a 5ml liquid LB test tube, and cultivate it at 37.0°C and 150r / min for 12h as the first-level seed;

[0107] Secondary seed preparation: Inoculate 5 ml of the cultured primary seeds into 500 ml of liquid LB medium, and cultivate them at 37.0° C. and 150 r / min for 12 hours as secondary seeds.

[0108] Shaker-induced expression:

[0109] Propagation of strains: Take 10ml of the primary seed solution and inoculate them into two 1000ml LB liquid medium respectively, shake and cultivate to OD at 220r / min 600 The nm value is 0.6-0.8.

[0110] Induction: Add IPTG (isopropyl-β-D-thiogalactopyranoside) inducer (fi...

Embodiment 2

[0113] Induction of Recombinant Escherichia coli BL21(DE3)-pET-30a-p27 in Fermenter

[0114] S101 strain culture:

[0115] Strain culture: Take a BL21(DE3)-pET-30a-p27 glycerol strain stored at -80°C, use an inoculation loop to dip the strain into four sections on the LB plate, and culture overnight at 37°C;

[0116] Preparation of first-level seed liquid: take the monoclonal cultured overnight in a 5ml liquid LB test tube, and cultivate it at 37.0°C and 150r / min for 10h as the first-level seed;

[0117] Secondary seed preparation: Inoculate 5 ml of the cultivated primary seeds into 500 ml of liquid LB medium, and cultivate for 10 hours at 37.0° C. and 150 r / min as secondary seeds.

[0118] S102 inoculation:

[0119] pH electrode calibration: wash the pH electrode with purified water, dry the filter paper and put it in a buffer solution with a pH of 6.86 for calibration, take out the pH electrode, wash the filter paper with purified water and dry it, then put it into a buffe...

Embodiment 3

[0129] Induction of Recombinant Escherichia coli BL21(DE3)-pET-30a-p27 in Fermenter

[0130] S101 strain culture:

[0131] Strain culture: Take a BL21(DE3)-pET-30a-p27 glycerol strain stored at -80°C, use an inoculation loop to dip the strain into four sections on the LB plate, and culture overnight at 37°C;

[0132] Preparation of first-level seed liquid: Take the monoclonal cultured overnight in a 5ml liquid LB test tube, and cultivate it at 37.0°C and 150r / min for 12h as the first-level seed;

[0133] Secondary seed preparation: Inoculate 5 ml of the cultured primary seeds into 500 ml of liquid LB medium, and cultivate them at 37.0° C. and 150 r / min for 12 hours as secondary seeds.

[0134] S102 inoculation:

[0135] pH electrode calibration: wash the pH electrode with purified water, dry the filter paper and put it in a buffer solution with a pH of 6.86 for calibration, take out the pH electrode, wash the filter paper with purified water and dry it, then put it into a bu...

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Abstract

The invention relates to the technical field of biotechnology, in particular to a method for efficiently expressing avian leukosis P27 protein and application of the method. The method comprises the following steps: culturing strains, inoculating, propagating, inducing expression, crushing and centrifuging, and drying to obtain the avian leukosis P27 protein. The method has the advantages that the avian leukosis P27 protein expression concentration is high, and the expression quantity is large. The yield of the avian leukosis P27 protein is high, and the expression quantity and the yield are stable. The production period is short; the cost is low; and the production efficiency is high. And the method has good stability and repeatability and is suitable for large-scale production.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a method for highly expressing avian leukemia P27 protein and its application. Background technique [0002] Avian leukemia is a general term for benign and malignant tumors of different tissues of poultry caused by avian leukosis virus (ALV). The virus belongs to the genus Alpharetrovirus of the Retroviridae family, and can be divided into 10 subgroups A to J. ALV mainly causes tumor death in infected chickens before and after sexual maturity. The infection rate and mortality rate vary, and the mortality rate can reach up to 20%. Although some chickens do not develop tumors after infection, they can cause decreased egg production performance and even immunosuppression. The control of this kind of disease is mainly to cut off the vertical transmission through population purification. Once the breeder flock is found to be positive for ALV, it should be eliminated in time t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K14/15G01N33/569C12R1/19
CPCC12N1/20C07K14/005G01N33/56983C12N2740/11022G01N2333/15
Inventor 王云峰王牟平高玉龙康志勇董明奇吴晓婵谢小明刘文韩庆安马宏伟顾文源
Owner 哈尔滨国生生物科技股份有限公司
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