Construction And Purification Of Therapeutic DNA Vaccine Against Human Hepatocellular Carcinoma

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 achi eve the purpose of tumor removal or control.Objective:To study the strategy of cooperative therapy of anti-cancer gene, two anti-tum-or gene, HBV X gene, MAGE-1 gene, were inserted into eukaryotic expressive plasmid vector to construct recombinant (pVAX-aHCC) and design experiment to study the anti-t umor effects of pVAXl-aHCC in vivo and ex vitro. The study established a solid founda-tion of cooperative cancer gene therapy, and has considerable significance in developing safe and/or effective anti-tumor medicine.Methods:The recombinant expression plasmid of pVAX-aHCC was constructed by inserti-ng HBV X gene and MAGE-1 gene into the downstream of prom-oter (CMV). The con s-tructed recombinant anti-tumor DNA was amplified in JM109 strain of Escherichia coli. After fermated in complex medium, the fermentation technology of pVAX-aHCC was e-st ablished. The Bacterium were collected through centrifugalization and then the plasmid w as prepurified by alkaline lysis, calcium chloride precipitation, PEG precipitation and soo n. 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 quail-ty and safety, several specifications were assessed by methods recommended by FDA, in-eluding supercoiled form percentage, transformation efficiency, identity, overall yield, puri-ty (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 liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed. (2) The result is as follows, pH 7.2±0. 2, supercoiled form percentage 91.7%, trasfection effiency 6.20×105 (cfu/μg plasmid),OD 260/280 1.79. Host protein, RNA and genomic DNA were undetectable. And the purified pl asmid was stable and can bear a certain high temperature. The result shows that the p-ur ification technology is feasible and it lays a basis for large-scale production of anti tu-m or therapeutic DNA vaccine as well as the others.Conclusion:In this study, a new type of liver cancer therapeutic recombinant DNA vacci-ne (pVAX-aHCC) was successfully constructed through the use of eukaryotic expressionp lasmid pVAX-1. And recombinant genetic vaccines werepurified by Sepharose 6FF, Plas- mid Select, DEAE Sepharose FF chromato graphy system. The purified plasmid DNA of high purity, consistent with FDA recommended using plasmid DNA of clinical quality sta-ndards.