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Plant expression vector and construction method thereof, and method for producing chicken alpha interferon by utilizing crowtoe as bioreactor

A plant expression vector and alpha interferon technology, applied in the field of producing medicinal protein chicken alpha interferon, can solve the problems of restricting the wide application of interferon, high cost, etc., to improve the immune function of the body, reduce the production cost, and achieve a long green grass period. Effect

Inactive Publication Date: 2011-07-13
GUIZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the clinical value of interferon is extremely high, the high cost of microbial fermentation and animal cell production seriously restricts the wide application of interferon in poultry industry

Method used

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  • Plant expression vector and construction method thereof, and method for producing chicken alpha interferon by utilizing crowtoe as bioreactor
  • Plant expression vector and construction method thereof, and method for producing chicken alpha interferon by utilizing crowtoe as bioreactor
  • Plant expression vector and construction method thereof, and method for producing chicken alpha interferon by utilizing crowtoe as bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of recombinant vector pSFRLH

[0051] (1) Materials and methods

[0052] 1. Plasmids and strains

[0053] Plasmid pbluescriptK (pBSK) was purchased from Shanghai Jierui Biological Company. Plasmid pSH737, Escherichia coli strain DH5α and Agrobacterium strain EHA105 have been publicly reported and can be purchased through the company or other public channels. Project highlights are available free of charge.

[0054] 2. Receptor material

[0055] The seeds of Leo japonicus (Lotus corniculatus L.) are sown after rubbing and scratching the seed coat with stone sand, and are managed in the field until the flowering period begins to be used.

[0056] 3. Plant expression vector construction

[0057] The ChIFN-α gene was designed and synthesized according to the chicken α-interferon sequence (GenBank No: U07868). The sequence contains KpnI / SalI and XhoI restriction enzyme sites, and the synthetic sequence was constructed on pBSK(+) to obtain the pBSK...

Embodiment 2

[0061] Embodiment 2: Agrobacterium-mediated transformation and regeneration of japonicus japonicus

[0062] (1) Bacterial liquid preparation

[0063] The fresh Agrobacterium liquid carrying the plant expression vector pSFRLH was inoculated in YEP liquid medium (additional Km100mg / L, rif20mg / L) at a ratio of 1:100, and cultured at 28°C for 8h (185rpm / min) with shaking until the logarithmic phase of growth, 4 Centrifuge (6500rpm / min) at ℃ for 15min and resuspend the bacteria (OD600=0.1-0.2) in MS plant liquid medium (added with 200μM As) for 2h before use.

[0064] (2) Infection transformation

[0065] Freshly picked branches and leaves of japonicus japonicus in full bloom stage on sunny days, rinsed with running water, soaked in 75% medical alcohol for 30s, sterilized with 0.1% mercuric chloride solution for 3min, rinsed with sterile water several times, the main stems, flower stems and After the young leaves were cut into sections or pieces, the prepared resuspended bacteria...

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Abstract

The invention relates to a plant expression vector and a construction method thereof, and a method for producing chicken alpha interferon by utilizing crowtoe as a bioreactor, belonging to the technical field of biology. The plane expression vector pSFRLH is shown as the figure 1 in the specification. In the invention, main stem sections, rachis stem sections and tender leaves of crowtoe are used as explants, and a constructed plane expression vector chicken alpha interferon gene ChIFN-alpha is subjected to genetic transformation and regeneration and then subjected to PCR (Polymerase Chain Reaction), RT-PCR (Reverse Transcription-Polymerase Chain Reaction), Southern blot, Western blot and ELISA (Enzyme-Linked Immuno Sorbent Assay). Proved by a detection result, the ChIFN-alpha gene is integrated into a crowtoe chromosome genome and correctly transcribed, translated and expressed, and recombinant protein can be neutralized by a ChIFN-alpha antibody. The invention lays the foundation for producing animal medical protein by utilizing crowtoe pasture grass as a bioreactor and provides a new thinking for the control of animal epidemic diseases.

Description

technical field [0001] The invention relates to the field of biological biotechnology transformation, in particular to a method for producing medicinal protein chicken alpha interferon using japonicus forage as a bioreactor. Background technique [0002] α interferon (Alpha interferon, IFN-α) is a kind of cytokine with powerful and broad-spectrum antiviral effect, and is an important component of animal defense system. Capsule found. Chicken alpha interferon (chIFN-α) on avian influenza (AIV), Newcastle disease (NDV), infectious bursitis (IBDV), infectious bronchitis (IBV), Marek's disease (MDV) And Rou's sarcoma (RSV) and other viruses have inhibitory effect. These viral diseases are important infectious diseases that endanger the development of the poultry industry, and often cause great economic losses. In recent years, chicken viral diseases represented by highly pathogenic avian influenza have even threatened human health. Although the clinical value of interferon is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/20C12P21/02A01H4/00
Inventor 赵德刚宋莉卢凌霄李岩
Owner GUIZHOU UNIV
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