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Melanoma therapeutic plasmid DNA (deoxyribonucleic acid) vaccine pSVK-CAVA preparation method as well as dedicated engineering bacterium and fermentation culture medium thereof

A DNA vaccine and fermentation medium technology, applied in the biological field, can solve the problems of difficult separation of plasmid DNA, poor therapeutic effect of open-loop and linearized plasmid DNA, etc.

Inactive Publication Date: 2013-07-24
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Application Information

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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  • Melanoma therapeutic plasmid DNA (deoxyribonucleic acid) vaccine pSVK-CAVA preparation method as well as dedicated engineering bacterium and fermentation culture medium thereof
  • Melanoma therapeutic plasmid DNA (deoxyribonucleic acid) vaccine pSVK-CAVA preparation method as well as dedicated engineering bacterium and fermentation culture medium thereof
  • Melanoma therapeutic plasmid DNA (deoxyribonucleic acid) vaccine pSVK-CAVA preparation method as well as dedicated engineering bacterium and fermentation culture medium thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0041] 1) Preparation of seed liquid: 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;

[0042] 2) Fermentation culture: The seed liquid was planted in a 5L fermenter (pilot production) according to the ratio 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 added 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

[0051] Example 1. Screening and Stability Detection of High Stability Host Bacteria of Melanoma Therapeutic Plasmid DNA Vaccine pSVK-CAVA

[0052] 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 respectively, and the transformation method was Hananhan method (Molecular Cloning Experiment Guide (Third Edition) 87 -92 pages), Inoue method (Molecular Cloning Experiment Guide (Third Edition) pages 93-96), calcium chloride method (Molecular Cloning Experiment Guide (Third Edition) pages 96-99), electric shock transformation method (Molecular Cloning Experiment Guidelines (Third Edition) pp. 99-102).

[0053] Take the conversion of XL10-Gold by calcium chloride method as an example to illustrate:

[0054] 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 repli...

Embodiment 2

[0061]Example 2, identification of melanoma therapeutic plasmid DNA vaccine pSVK-CAVA engineering bacteria E.coliXL10-Gold / CAVA

[0062] 1. Detection of genetic stability and structural stability of the working seed bank

[0063] One working seed was revived by heating in a water bath at 37°C, inoculated into 5 mL of LB medium, cultured at 37°C and 200 rpm for 12 hours, and then subcultured at a ratio of 1:100. This method was continuously passed down for 30 generations. 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.

[0064] Results The engineering bacteria E.coli XL10-Gold / CAVA was continuously subcultured for 30 generations, and the plasmid was extracted every 5 generations. The 1% agarose gel electrophoresis pattern of the plasmid (see figure 2 ) showed that the engineered bacteria exhibited good genetic ...

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Abstract

The invention discloses a melanoma therapeutic plasmid DNA (deoxyribonucleic acid) vaccine pSVK-CAVA preparation method and a dedicated engineering bacterium and a fermentation culture medium thereof. The engineering bacterium is Escherichia coli XL10-Gold which contains converted melanoma therapeutic plasmid DNA pSVK-CAVA and is named as E.coli XL10-Gold / CAVA. Each 1L of high-yield fermentation culture medium comprises: 12g of casein peptone, 16.61g of yeast extract, 3.05mL of glycerol, 1g of NH4Cl, 100mL of mixed solution of 0.17mol / L of KH2PO4 and 0.72mol / L of K2HPO4, 0.185g of MgSO4, and 1mL of trace elements. The engineering bacterium is high in stability, can be subjected to continuous passage for more than 30 times, can meet the demand of plasmid DNA vaccine pSVK-CAVA pilot test process, the plasmid volume yield obtained after fermentation of the high-yield culture medium is high, and the engineering bacterium can be used for fermentation production of the melanoma therapeutic plasmid DNA vaccine pSVK-CAVA.

Description

technical field [0001] 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 [0002] 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 progress. . Plasmid DNA vaccines have the characteristics of high safety, no carrier immunogenicity, and easy production. Therefore, compared with live virus and virus-based vaccines, they have unique advantages. Hundreds of vaccines have entered the clinical trial stage. At present, 4 kinds of plasmid DNA vaccines and produ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C40B50/06C40B40/02A61K48/00A61P35/00C12R1/19
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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