Preparation method of lung cancer resistant plasmid T-VISA (VP16-Gal4-WPRE integrated systemic amplifier)-Bik (bcl-2interacting killer) DD

Abstract

The invention provides a preparation and purification method of a lung cancer resistant plasmid T-VISA (VP16-Gal4-WPRE integrated systemic amplifier)-Bik (bcl-2interacting killer) DD. The method comprises the steps as follows: seed amplification culture, fermented cultivation, centrifugal collection of thalli, thallus pyrolysis, supernatant collection and vacuum filtration, sieve chromatography, affinity chromatography, ion chromatography, isopropyl alcohol precipitation, PBS (phosphate buffer) dissolution and the like. With the adoption of the method, the fermentation content of the finally obtained lung cancer resistant plasmid T-VISA-BikDD is 150-200 mg / L, and the superhelix proportion of the plasmid after purification is higher than 90%.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mass production and purification method of an anti-lung cancer T-VISA-BikDD plasmid. Background technique [0002] The significance and demand of plasmid DNA as a biological agent (especially as a carrier of gene therapy and gene vaccine) is increasing. In recent years, the use of recombinant plasmids in gene therapy has been extensively and deeply studied. At present, most tumor gene therapy uses cytomegalovirus (cytomegalovirus, CMV) promoter lacks tissue specificity. Some researchers used tumor-specific promoters instead of CMV for gene therapy research, and found that the activity of tumor-specific promoters was extremely low (generally only about 1% of that of CMV promoters), and its curative effect was limited. Aiming at the scientific problem of low activity of tumor-specific promoters, Professor Xie Xiaoming's team at Sun Yat-sen University Cancer Hospital developed a tumo...

AI Technical Summary

Problems solved by technology

At present, many pharmaceutical companies at home and abroad are establishing large-scale plasmid DNA production processes that meet pharmaceutical specifications, such as batch fermentation, purification and other large-scale preparation links, to meet the growing clinical needs, but there are still some problems in these production processes. Insurmountable bottlenecks, such as vector construction, bacterial plasmid copy number in fermentation, cell lysis, bacterial chromosomal DNA removal during purification, bacterial endotoxin removal, final product quality control, etc.

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