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Expression of P27 protein and its use

A technology of p27 and protein, which is applied in the expression of p27 protein and its application field, which can solve the problems of cumbersome avian leukosis virus, high cost of plasma poisoning, complicated operation, etc.

Inactive Publication Date: 2005-05-18
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The reproduction of avian leukosis virus is a rather cumbersome work, and there is no specific applicable cell line for the reproduction of the virus
Whether the preparation method is to use cells to reproduce or inoculate chickens to prepare plasma toxin, there are problems such as high cost, low yield, time-consuming, complicated operation, and low purity.

Method used

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  • Expression of P27 protein and its use
  • Expression of P27 protein and its use
  • Expression of P27 protein and its use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 P27 protein preparation process

[0024] 1 Preparation of recombinant engineered bacteria

[0025] A recombinant engineering bacterium for expressing p27 protein was constructed, the expression vector used was pET28α, and the host bacterium was Escherichia coli BL21(DE3).

[0026] (1) Construction process of recombinant engineered bacteria

[0027] ① Preparation of the target fragment to be inserted

[0028] a) Double digestion of recombinant plasmid pGEM-Teasy / p27:

[0029] The recombinant plasmid pGEM-Teasy / p27 was double digested with BmaHI and SalI, and the reaction system was as follows:

[0030] The total reaction volume is 50 μl

[0031] BmaHI 2 μl

[0032] SalI 2μl

[0033] 10×T buffer 7.5μl

[0034] Substrate DNA 20μl

[0035] Sterilized water 18.5μl

[0036] The above components were mixed evenly, and after brief centrifugation, incubated at 37° C. for 6 hours.

[0037] b) recovery of enzyme digestion products:

[0038] Agarose gel electro...

Embodiment 2

[0137] Example 2 Induced expression of p27 gene and purification of expression product

[0138] 1. Induced expression of p27 gene:

[0139] (1) Expression process

[0140] ①Select a well-growing positive single colony and inoculate it in 3ml LB (containing 30μg / ml Kana) medium, culture overnight at 37°C.

[0141] ② Take out 0.05ml and inoculate it into 5ml LB (containing 30μg / ml Kana) medium, and incubate at 30°C for 2-3 hours (OD600 to 0.4-0.6).

[0142] ③Add IPTG to a final concentration of 3mM, and induce expression at 30°C for 5 hours.

[0143] ④ Centrifuge at 5000rpm at 4°C for 10 minutes, discard the culture medium, collect the bacterial pellet, and store at -20°C.

[0144] (2) Reagents used for expression

[0145] ①Kanamycin (Kana) stock solution (30mg / ml): 300mg kanamycin was dissolved in 10ml sterilized water, sterilized by filtration, and dispensed at -20°C for use.

[0146] ② LB liquid culture medium Tryptone 10g, yeast extract 5g, NaCl 10g, dissolved in 950ml ...

Embodiment 3

[0242] Example 3 Preparation of rabbit anti-p27 antibody

[0243] (1) Immune acquisition of rabbit anti-P27 antibody

[0244] Rabbits were immunized with the prepared purified p27 protein by subcutaneous injection, intradermal injection and intramuscular injection.

[0245] 1. First immunization: Intradermal and intramuscular injection, the injection dose is 100μg / monkey. Take a certain amount of antigen solution, add an equal amount of Freund's complete adjuvant, emulsify completely, and inject 50 μg intradermally and intramuscularly.

[0246] 2. Second immunization: One week later, subcutaneous and intramuscular injections are used for the second immunization, and the injection dose is 200 μg / monkey. Take a certain amount of antigen solution, add the same amount of Freund's incomplete adjuvant, emulsify completely, and inject 100 μg subcutaneously and intramuscularly into each rabbit.

[0247] 3. Three immunizations: One week after the second immunization, the third immuniz...

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Abstract

The present invention relates to prokaryotic expression of fowl leukemia related p27 protein and its application. The present invention constitutes new recombinant expression plasmid with the designed specific primer, so as to express high level and high purity p27 protein successfully in prokaryotic cell.

Description

technical field [0001] The present invention relates to the expression of avian leukosis virus P27 protein and the application of the protein. Background technique [0002] Avian Leukosis is a general term for a variety of tumor diseases in poultry caused by viruses in the avian leukosis virus and poultry sarcoma virus group [1] . Avian leukemia is a common disease in poultry. Currently there is no vaccine and no effective antiviral drugs. Purification measures are often used to cultivate leukemia-free chickens. [0003] Avian leukosis virus can spread vertically or horizontally, causing great harm to the production of poultry industry. Several kinds of poultry embryo-derived vaccines and poultry embryo-derived cell vaccines produced by domestic veterinary and human medical biological product factories all use chicken embryos as raw materials. Through the detection of avian leukosis antigen, the spread and reinfection of the virus are caused, and the harm can be imagined. ...

Claims

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Application Information

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IPC IPC(8): C07H21/04C07K14/005C12N15/33C12N15/70G01N33/68
Inventor 陈福勇
Owner CHINA AGRI UNIV
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