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Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha

A technology of bovine interferon and alpha gene, which is applied in the preparation method of peptide, interferon, cytokine/lymphokine/interferon, etc.

Inactive Publication Date: 2015-04-22
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Recombinant bovine interferon-α is rarely reported

Method used

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  • Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha
  • Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha
  • Primers for obtaining genes of bovine interferon alpha and preparation method for recombinant bovine interferon alpha

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] A preparation method of recombinant bovine interferon alpha, comprising the following steps:

[0064] a. obtaining the bovine interferon alpha gene, comprising the following steps:

[0065] The RNA extraction kit from TAKARA company was used to extract RNA from bovine liver tissue, and the primers were used for RT-PCR reaction;

[0066] Primer sequence: Upstream: ATA GGA TCC TGC CAC CTGGCCT CAC

[0067] Downstream: ATA AAG CTT TCA GTC CTT TCT CCT G

[0068] The primers have BamHI and HindIII restriction sites respectively;

[0069] The target gene of bovine interferon α is:

[0070] tgccacctgc ctcacaccca cagcctggcc aacaggaggg tcctgatgct cctgcaacaa 60

[0071] ctgagaaggg tctccccttc ctcctgcctg caggacagaa atgacttcga attcctccag 120

[0072] gaggctctgg gtggcagcca gttgcagaag gctcaagcca tctctgtgct ccacgaggtg 180

[0073] accccagcaca ccttccagct cttcagcaca gagggctcgc ccgccacgtg ggacaagagc240

[0074] ctcctggaca agctacgcgc tgcgctggat cagcagctca ctgacctgca agcctgtctg 300

...

Embodiment 2

[0101] Identification of Recombinant Bovine Interferon-α:

[0102] Protein Quantitative Detection:

[0103] The Lowry method was used to measure the standard protein of the China Institute for the Control of Food, Drug and Biological Products, and the protein content was determined to be ≥ 1.0 mg / ml.

[0104] SDS-PAGE electrophoresis detection:

[0105] Compared with the empty bacterium control, the recombinant bacterium has a densely stained new target protein band at about 35KD.

[0106] Western Blot results:

[0107] Abcam mouse anti-bovine interferon (1:2000 dilution) was used as polyclonal antibody, and goat anti-mouse IgG-HRP was used as secondary antibody (1:5000 dilution). Recombinant bovine interferon α samples can specifically react with anti-bovine interferon polyclonal antibody, and a specific band appears at about 35KD.

Embodiment 3

[0109] Recombinant bovine interferon α titer detection: According to the microcytopathic inhibition method, using the Hep-2 cell / VSV virus system: culture Hep-2 cells with DMEM nutrient solution on a 96-well microcell culture plate, and place in 5% CO 2 Cultivate in an incubator at 37°C for 24 hours, add different doses of recombinant bovine interferon α, discard after 24 hours, and inoculate 100TCID respectively 50 VSV virus. The results showed that the obtained recombinant bovine interferon α had obvious inhibitory effect on the cytopathic effect caused by VSV. After the untreated cells were inoculated with the virus, the cells became round, shed, and disintegrated. After the obtained recombinant bovine interferon α-treated cells were inoculated with the virus, they were continuously observed under an inverted microscope, and the cell morphology was normal without any pathological changes, and the measured titer was >1.0×10 6 IU / ml.

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Abstract

The invention discloses primers for obtaining genes of bovine interferon alpha and a preparation method for recombinant bovine interferon alpha. The preparation method comprises the following steps: a, obtaining genes of bovine interferon alpha; b, constructing a cloning vector; c, constructing an expression vector; d, expressing recombinant protein; e, preparing a recombinant bovine interferon alpha crude product; and f, purifying the bovine interferon alpha crude product. Compared with the prior art, the bovine interferon alpha is a cell factor capable of inducing bovine cells to generate various broad-spectrum antiviral proteins and can be used for treating diseases caused by bovine vesicular stomatitis virus, foot and mouth disease virus, bovine viral diarrhea virus, bovine respiratory syncytial virus, and the like.

Description

technical field [0001] The invention belongs to the technical field of gene recombination, and in particular relates to a primer for obtaining bovine interferon-α gene and a method for preparing recombinant bovine interferon-α. Background technique [0002] With the rapid development of the global livestock and poultry breeding industry in recent years, my country's cattle industry has developed from a sideline business in rural households to a major industry in society. Both the variety and quantity of animals are in the forefront of the world. However, there are still many problems in animal husbandry, such as vesicular stomatitis virus, foot-and-mouth disease virus, bovine viral diarrhea virus, bovine respiratory syncytial virus and other infectious diseases, which will break out in different seasons every year. Once these animals are infected, The characteristics of acute onset, severe clinical symptoms, easy transmission and high mortality rate have caused huge economic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/70C07K14/56C07K1/22C07K1/18C07K1/16
Inventor 王明丽赵俊李树启
Owner ANHUI JIUCHUAN BIOTECH
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