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Production process of human lysozyme as AIDS treating medicine with plant as bioreactor

A human lysozyme, fusion protein technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve the problems of difficulty in the source of human lysozyme, no industrial production, etc., to facilitate the detection of transformed plants Effect

Inactive Publication Date: 2005-06-01
甘肃亚盛盐化工业集团有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, due to the difficulty in the source of human lysozyme, there is no industrial production in the world at present
Using plants as a reactor to produce human lysozyme as an anti-AIDS drug has not been reported at home and abroad so far

Method used

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  • Production process of human lysozyme as AIDS treating medicine with plant as bioreactor
  • Production process of human lysozyme as AIDS treating medicine with plant as bioreactor
  • Production process of human lysozyme as AIDS treating medicine with plant as bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1. Expression of recombinant human lysozyme cDNA in Escherichia coli

[0023] 1. Construction of Escherichia coli expression vector of recombinant human lysozyme cDNA:

[0024] (1) Cloning of lysozyme cDNA: total mRNA was extracted from human placenta and subjected to RT-PCR reaction. Primers (synthesized by Beijing Aoke Bioengineering Co., Ltd.) were designed according to the lysozyme gene sequence (HSU25677) published by Genebank:

[0025] Primer 1: 5'-GCG GCTAGC ATGAAGGCTCTCATTGTTCTG-3'

[0026] Primer 2: 5'-GCG CTCGAG GCTTACACTCCACAACCTTGA-3'

[0027] A cDNA fragment encoding the mature protein of human lysozyme was obtained.

[0028] (2) Construct the cDNA fragment of the human lysozyme mature protein into pET-28a (product of Novegon Company) containing the Lac promoter: first digest the cDNA fragment of the human lysozyme mature protein with Nco I / EcoR I, and connect it into In Escherichia coli expression plasmid pET-28a which was also digested ...

Embodiment 2

[0044] Embodiment 2. Construction of the plant expression vector of human lysozyme gene

[0045] In this example, the gene encoding human lysozyme was linked downstream of the strong plant nuclear promoter 35S, and there was an enhancing Ω sequence between 35S and the gene, which could enhance the gene expression after transformation. In the constructed plant transformation vector, the expression box of the screening marker gene and the expression box of the marker gene green fluorescent protein gene (GFP) are included at the same time.

[0046] A. Construction of plant expression vector pBlyzg for gene gun transformation

[0047] 1. Plasmid pUC-18 was double digested with BamHI and SalI, and the cut linear plasmid was recovered by electrophoresis.

[0048] 2. The plant nuclear transformation plasmid pBTu (using pBluescript SK(+) as the basic plasmid, constructs the required multiple cloning site, and contains the plant nuclear strong promoter 35S, the Ω sequence that plays a...

Embodiment 3

[0056] Embodiment 3. Obtaining of transgenic plants

[0057] 1. Agrobacterium transformation method to introduce lysozyme gene into lettuce

[0058] A: Transform the plasmid pBGlyzg into Agrobacterium

[0059] (1) Preparation of competent cells of Agrobacterium LBA4404:

[0060] A single colony of Agrobacterium tumefaciens LBA4404 was picked and inoculated in 5 ml of YEP (containing 20 mg / L rifampicin, 30 mg / L streptomycin) medium, and cultured overnight at 28° C. with shaking at 200 rpm.

[0061] Add 2ml of the overnight cultured bacterial solution to 50ml of YEP medium containing the same antibiotic, shake quickly at 220rpm for 3-4h at 28°C, and make the OD 600 = 0.5.

[0062] Centrifuge at 5000rpm for 5min, remove the supernatant, add 10ml of 0.15mol / L NaCl to suspend the cells, and ice-bath for 20min.

[0063] Centrifuge at 5000rpm for 5min at 4°C, remove the supernatant, and add 1ml of pre-cooled 20mmol / L CaCl 2 Resuspend the cells (15% glycerol), aliquot into 1.5ml Ep...

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Abstract

The present invention provides production process of human lysozyme as AIDS treating medicine with plant as bioreactor. Human lysozyme gene is cloned from human placenta and constructed into prokaryotic expression vector for efficient expression. Plant expression vector is constructed in lettuce, and the human lysozyme gene is introduced into lettuce nuclear genome to realize the efficient expression of human lysozyme gene in higher plant and to obtain one kind of lettuce containing human lysozyme. Human lysozyme mass produced in the said method may be used in treating AIDS, tumor and viral diseases.

Description

1. Technical field: [0001] The invention relates to a protein preparation method. Specifically, it relates to a method for producing human lysozyme protein using plants as "bioreactors". 2. Background technology: [0002] AIDS (AIDS, acquired immunodeficiency syndrome) is an infectious disease caused by human immunodeficiency virus (HIV, human immunodeficiency virus). After HIV enters the human body, it will take several years, even a latent period of 10 years or longer before the onset of the disease. HIV severely damages the immune function of the human body, and the patient is repeatedly infected with various diseases due to the extremely reduced ability to resist the disease, such as herpes zoster, oral fungal infection, tuberculosis, enteritis, pneumonia, encephalitis and other infections caused by special pathogenic microorganisms. Malignant tumors often occur. Finally, due to long-term exhaustion of physical strength, he died of systemic failure. According to stat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H1/00A61K38/43A61K38/47C12N9/36C12N15/56C12N15/60C12N15/62C12N15/63C12N15/79C12P21/02
Inventor 周长生谢小冬陈正华
Owner 甘肃亚盛盐化工业集团有限责任公司