Study On Purification Of Therapeutic DNA Vaccine Against Human Lung Cancer

Background:Anti-cancer (DNA vaccines) is a research hotspot in recent years at home and abroad, It is mainly by activating the patient's own immune system in order to achieve the purpose of tumor removal or control.Objective:The study established a solid foundation of cooperative cancer gene therapy, and has considerable significance in developing safe and/or effective anti-tumor medicine.Methods:The recombinant expression plasmid of PVNY-ESO-1SSX-2 was constructed by inserting NY-ESO-1 gene and SSX-2 gene. The constructed recombinant anti-tumor DNA was amplified in JM109 strain of Escherichia coli. After fermated in complex medium, the fermentation technology of PVNY-ESO-1 SSX-2 was established. The Bacterium was collected through centrifugalization and then the plasmid was pre-purified by alkaline lysis, calcium chloride precipitation, PEG precipitation and so on. Then the plasmid was applied to a Sepharose 6FF, Plasmid Select, DEAE Sepharose FF column, and crude product was successfully isolated. When it comes to plasmid quality and safety, several specifications were assessed by methods recommended by FDA, including supercoiled form percentage, transformation efficiency, identity, overall yield, and purity (OD260/280)-Residual impurities levels such as RNA, host protein, host genomic DNA in final product were also detected.Results:(1) In this study, a new type of lung cancer therapeutic recombinant DNA vaccine (PVNY-ESO-1 SSX-2) was successfully constructed. (2) The ratio of A260 to A280 was 1.85-1.92. The content of circular DNA was more than 93%. The end toxin and host DNA contents were little, less than 10EU/mg. Host protein, RNA and genomic DNA were undetectable. The result shows that the purification technology is feasible and it lays a basis for large-scale production of anti-tumor therapeutic DNA vaccine as well as the others.Conclusion:In this study, a new type of lung cancer therapeutic recombinant DNA vaccine (PVNY-ESO-1SSX-2) was purified by Sepharose 6FF, Plasmid Select, DEAE Sepharose FF chromatography system. The purified plasmid DNA of high purity, consistent with FDA recommended using plasmid DNA of clinical quality standards.