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Fermentation medium and method for producing plasmid DNA vaccine pSVK-CAVA for treatment of melanoma by fermentation medium

A DNA vaccine and fermentation medium technology, applied in the field of fermentation medium and the production of melanoma therapeutic plasmid DNA vaccine pSVK-CAVA, can solve the problem that the therapeutic effect of open-loop and linearized plasmid DNA is not good, and the plasmid DNA is difficult to separate, etc. question

Inactive Publication Date: 2014-07-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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AI Technical Summary

Problems solved by technology

The FDA believes that the therapeutic effect of open-circle and linearized plasmid DNA is not as good as that of supercoiled plasmid DNA (Food and Drug Administration (FDA), Guidance for industry: considerations for plasma deoxyribonucleic acid vaccines for infectious disease indications, 2007, http: / / www.fda .gov / cber / gdlns / plasdnavac.htm.), however, several forms of plasmid DNA are difficult to separate during the purification process, so how to obtain a high proportion of supercoiled plasmid DNA should be optimized during fermentation (Ying C, Rodriguez S, Hebel H. DNA vaccine manufacture: scale and quality. Expert Reviews Vaccines, 2009, 8(9): 1277-1291.)

Method used

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  • Fermentation medium and method for producing plasmid DNA vaccine pSVK-CAVA for treatment of melanoma by fermentation medium
  • Fermentation medium and method for producing plasmid DNA vaccine pSVK-CAVA for treatment of melanoma by fermentation medium
  • Fermentation medium and method for producing plasmid DNA vaccine pSVK-CAVA for treatment of melanoma by fermentation medium

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0042] 1) Preparation of seed solution: Resuscitate one E.coli XL10-Gold / CAVA working seed from -70°C, inoculate it in 5 mL of LB liquid medium at a ratio of 1:100, and incubate at 37°C and 200 rpm for 12 hours. Transfer to 100mL LB liquid medium according to the ratio of 1:100, culture at 37°C and 200rpm for about 12 hours, and obtain seed liquid at this time;

[0043] 2) Fermentation culture: The seed liquid was planted in a 5L fermenter (pilot production) according to the amount of 1:100 for fermentation culture. The amount of high-yield fermentation medium ITB contained in the fermenter was 3L, and the pH value was controlled at 7.0±0.1 (Automatically add H through the fermenter 2 SO 4 and NH 3 .H 2 0 to adjust the pH value of the medium), the culture temperature was 37°C, and the dissolved oxygen was controlled at about 30% (when the dissolved oxygen decreased to this value, it was adjusted by supplementing pure oxygen and increasing the stirring speed). After 6 hours...

Embodiment 1

[0052] Example 1. Screening and Stability Detection of High Stability Host Bacteria of Melanoma Therapeutic Plasmid DNA Vaccine pSVK-CAVA

[0053] The melanoma therapeutic plasmid DNA vaccine pSVK-CAVA plasmid DNA was transformed into different genotypes of Escherichia coli competent cells DH5α, DH10β, Top10 and XL10-Gold, and the transformation method was the Hananhan method (Molecular Cloning Experiment Guide (Third Edition) 87 -92 pages), Inoue method (Molecular Cloning Experiment Guide (Third Edition) 93-96 pages), calcium chloride method (Molecular Cloning Experiment Guide (Third Edition) 96-99 pages), electric shock transformation method (Molecular Cloning Experiment Guidelines (Third Edition) pp. 99-102).

[0054] Take the conversion of XL10-Gold by calcium chloride method as an example to illustrate:

[0055] The pSVK-CAVA plasmid DNA can be found in literature (Zhang Liang, Yan Jinqi, Wang Yue, et al. Construction and in vivo and in vitro expression of replicable ant...

Embodiment 2

[0062] Example 2, identification of melanoma therapeutic plasmid DNA vaccine pSVK-CAVA engineering bacteria E.coliXL10-Gold / CAVA

[0063] 1. Detection of genetic stability and structural stability of the working seed bank

[0064] Resuscitate 1 working seed by heating in a water bath at 37°C, inoculate in 5mL LB medium, culture at 37°C and 200rpm for 12 hours, then subculture at a ratio of 1:100. Plasmids were extracted from the strains of the 1st, 15th, 20th, 25th, and 30th generations, and the structural stability of the working seed bank was detected by 1% agarose gel electrophoresis and enzyme digestion.

[0065] Results Engineering bacteria E.coli XL10-Gold / CAVA was continuously subcultured for 30 generations, and the plasmid was extracted every 5 generations, and the 1% agarose gel electrophoresis pattern of the plasmid (see figure 2 ) showed that the engineered bacteria exhibited good genetic stability, the plasmid copy number remained basically stable, and at the same ...

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Abstract

The invention discloses a fermentation medium and a method for producing plasmid DNA vaccine pSVK-CAVA for the treatment of melanoma by the fermentation medium. The high-yield fermentation medium per 1L comprises 12g of casein peptone, 16.61g of yeast extract, 3.05mL of glycerol, 1g of NH4Cl, 100mL of a mixed solution of 0.17mol / L KH2PO4 and 0.72mol / L K2HPO4, 0.185g of MgSO4 and 1mL of trace elements. The plasmid with a high volumetric yield can be obtained by an engineered bacteria E. coli XL10-Gold / CAVA working seed and the fermentation medium and the fermentation production of DNA vaccine pSVK-CAVA for the treatment of melanoma can be achieved.

Description

[0001] The application date is April 16, 2013, the application number is 201310131566.3, and the title of the invention is a divisional application of the patent application entitled "Preparation Method of Melanoma Therapeutic Plasmid DNA Vaccine pSVK-CAVA and Its Special Engineering Bacteria and Fermentation Medium" . technical field [0002] The invention belongs to the preparation method of a plasmid DNA vaccine in the field of biotechnology, its special engineering bacteria and a high-yield fermentation medium, and in particular relates to a preparation method of a melanoma therapeutic plasmid DNA vaccine pSVK-CAVA, its special engineering bacteria and its high-yield fermentation Medium. Background technique [0003] Plasmid DNA vaccine is a new vaccine with great development potential discovered by research in recent years. Plasmid DNA vaccine has attracted the attention of scientists in the early 1990s. It has been developed for nearly 30 years and has made great progr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/85A61K48/00A61P35/00C12R1/19
Inventor 于继云阎瑾琦王宇徐元基张巍吴昊张亮朱晓明
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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