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Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha

A technology of alpha interferon and transgenic cell lines, applied to the optimized gene encoding chicken alpha interferon and its application in the preparation of chicken alpha interferon, which can solve undiscovered problems

Active Publication Date: 2011-01-19
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chicken and other poultry IFNs are similar to mammalian IFNs, and are also divided into type I IFN and type II IFN. It has been found that chicken type I IFN includes IFN-α and IFN-β, and type II includes IFN-γ. Discovery of IFN-ω and IFN-τ

Method used

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  • Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha
  • Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha
  • Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Discovery of Chicken ChIFNα-1 Gene

[0026] 1. Extraction of chicken total RNA

[0027] According to the DNA extraction kit of Invitrogen, chicken genomic DNA was extracted from the whole blood of chicken (the breed is Qingjiao Maji, purchased from Changping Animal Experimental Base of Beijing Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences).

[0028] 2. Design of specific primer pairs

[0029] Referring to the existing chicken interferon alpha gene sequence, the primer pairs are designed as follows:

[0030] Upstream primer: 5’-GGAATTC CATATG TGCAACCACCTTCGCC-3' (the NdeI restriction site is underlined);

[0031] Downstream primer: 5’-CCG GAATTC CTAAGTGCGCGTGTTGC-3' (EcoRI restriction site is underlined).

[0032] 3. Amplification of ChIFNα gene

[0033] Using the genomic DNA of step 1 as a template, PCR amplification is performed with the specific primer pair designed in step 2 to obtain PCR amplification products.

[0034]...

Embodiment 2

[0036] Example 2. Expression of ChIFNα-1 gene and ChIFNα-2 gene

[0037] 1. Preparation of ChIFNα-1 gene and ChIFNα-2 gene

[0038] 1. Preparation of ChIFNα-1 gene

[0039] Prepare the DNA shown in Sequence 2 of the Sequence Listing, use it as a template, and perform PCR amplification to obtain a PCR amplification product (ChIFNα-1 gene).

[0040] The primer pairs for PCR amplification are as follows:

[0041] Upstream primer: 5’-GGAATTC CATATG TGCAACCACCTTCGCC-3' (the NdeI restriction site is underlined);

[0042] Downstream primer: 5’-CCG GAATTC CTAAGTGCGCGTGTTGC-3' (EcoRI restriction site is underlined).

[0043] The PCR conditions were: 95°C for 5 minutes; 95°C for 30s, 55°C for 30s, 72°C for 50s, 30 cycles; 72°C for 10 minutes.

[0044] Carry out electrophoresis on the PCR amplified product of step 2 and see the result of electrophoresis figure 2 . figure 2 In the arrow, the target gene is about 500bp, which is consistent with the actual size (492bp).

[0045] 2. Preparation of ChI...

Embodiment 3

[0083] Example 3. Expression of ChIFNα-1 gene and chiIFNα-2 gene

[0084] 1. Preparation of recombinant bacteria and control bacteria

[0085] The pET28b-ChIFNα-1 prepared in step 2 of Example 2 was transformed into E. coli BL-21 to obtain recombinant bacteria. The pET28b-ChIFNα-2 prepared in step 2 of Example 2 was transformed into E. coli BL-21 to obtain a control bacteria.

[0086] 2. ChIFNα-1 gene and ChIFNα-2 gene expression

[0087] 1. Expression of ChIFNα-1 gene

[0088] ① Inoculate the recombinant bacteria in 3ml of LB liquid medium containing kanamycin, culture it overnight at 37°C for activation.

[0089] ②According to the volume ratio of 1:100, the activated bacteria were inoculated into 5ml LB medium containing kanamycin, cultured at 37°C for about 2h to the logarithmic growth phase (D 600nm The value reaches 0.5-0.8), a small amount is taken as an uninduced control, and the remaining part is added with an IPTG inducer (final concentration of 1mmol / L), and the expression is...

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Abstract

The invention discloses an optimization gene for coding chicken interferon alpha and application thereof in preparing the chicken interferon alpha. The optimization gene is DNA (deoxyribonucleic acid) shown as the sequence 2 in a sequence table. The expression ability of the gene is far higher than that of the traditional gene; the gene is inserted into the polyclone site of pET28b(+) to obtain a recombinant plasmid, and the recombinant plasmid is imported into escherichia coli BL21 to obtain a recombination strain. A large amount of ChIFN alpha protein can be prepared through carrying out simple induction culture on the recombination strain. The gene, the recombinant plasmid and the recombination strain has economic value for the production of the chicken interferon alpha and also has great value for broiler culture industry.

Description

Technical field [0001] The invention relates to an optimized gene encoding chicken alpha interferon and its application in preparing chicken alpha interferon. Background technique [0002] Interferon (Interferon, IFN) was first discovered in 1957 by British scientist Isaacs when studying the interference phenomenon of avian influenza virus. It is a glycoprotein with broad-spectrum antiviral activity and is stimulated by viruses and other types of interferon inducers. Produced by endothelial cells, macrophages, lymphocytes and somatic cells, it has anti-virus, anti-tumor, immune regulation and differentiation induction activities. The effect of interferon does not directly act on the virus, but stimulates cells to produce a variety of broad-spectrum antiviral proteins, which can exert antiviral effects through direct or indirect means. [0003] According to different structures and receptors, IFN can be divided into two types: type I IFN and type II IFN. Mammalian type I IFN mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/21C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C07K14/56C12P21/00C12R1/19
Inventor 刘文军瞿洪仁李晶杨利敏贾晓娟
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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