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

A technology of human lysozyme and gene, applied in botany equipment and methods, biochemical equipment and methods, pharmaceutical formulations, etc., can solve the problems of difficult source of human lysozyme, non-industrial production, etc., and achieve convenient detection of transformed plants Effect

Inactive Publication Date: 2004-01-21
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 2

[0042] With reference to the method detection such as Chen Fengzhen (Guangdong Silk Communication, 1988 (3): 35-42) the recombinant protein expressed by pET-lyz, pET-lyzg, the substrate is the bacterium powder of Micrococcus lyticus of Sigma Company, with 0.1MPH6. 2 Phosphate buffer solution is made into suspension, and standard enzyme solution is egg white lysozyme (43000 units / mg protein) of Sigma Company, according to formula lysozyme activity (U / mg)=OD450nm / (0.001*mg enzyme / ml reaction Liquid) to calculate the lysozyme activity, the average lysozyme activity recorded is 41500 units / mg protein, which can be used for medicinal purposes completely. Embodiment 2. Construction of the plant expression vector of human lysozyme gene

[0043] 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...

Embodiment 3

[0052] 2. Using T4 DNA ligase to connect the recovered PBG vector fragment and the recombinant human lysozyme gene fragment to obtain the expression vector plasmid PBGlyzg for biolistic transformation of the recombinant human lysozyme gene. Embodiment 3. Obtaining of transgenic plants

[0053] 1. Agrobacterium transformation method to introduce lysozyme gene into lettuce

[0054] A: Transform the plasmid pBGlyzg into Agrobacterium

[0055] (1) Preparation of competent cells of Agrobacterium LBA4404:

[0056] 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.

[0057]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.

[0058] Centrifuge at 5000rpm for 5min, remove the supernatant, add 10ml o...

Embodiment 4

[0102] The gene gun is the PDS-1000 / He Biolistic particle delivery system produced by the American BIO-RAD company. The parameters used are: vacuum degree 28 inches.Hg, bombardment distance 9cm, helium pressure 1100psi, for conversion. After the bombardment, the explants were placed on ordinary MS medium for 2 days, and then transformed into culture medium containing kanamycin. First, MB2 medium was used. After about 2-4 weeks of induction and selection culture, or When the callus appeared on the implant, it was transferred to MB5 medium and continued to cultivate. After three weeks, the obtained resistant green bud clusters were transferred to MB6 plus kanamycin for selection and differentiation medium, and continued on this medium. Twice, three weeks each time. The green shoots obtained are then transferred to MB 1 Add the differentiation medium of kanamycin, the regenerated seedlings growing up on this medium are transferred to the rooting medium of MN added kanamycin, an...

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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 Applications(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
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