Study On Construction And Purification Of Therapeutic DNA Vaccine Against Tumor

Malignant tumor is one of the most severe diseases of human being currently. The effectively treatment or control of tumor is imminent because its disease incidence and case fatality have a tendency of ascensus year by year. With the rapid development of molecular biology, genetic engineering and tumor immunology, cancer gene therapy has been seen as one of most possible means to cure tumor. Current methods of cancer gene therapy have some anti-tumor effects theoretically, however, the therapeutic effect in vivo is less obviously than in vitro. Tumor progress is a multi-factor, multi-stage and multi-gene process, to cure cancer, the conjoined application of anti-cancer gene is thought to be more effectively than single application o. To study the strategy of cooperative therapy of anti-cancer gene, three anti-tumor gene, apoptin VP3 gene, NDV HN gene and IL-18 gene, were inserted into eukaryotic expressive plasmid vector to construct recombinant (pVVP3IL-18HN) and design experiment to study the anti-tumor effects of pVVP3IL-18HN in vivo and in vitro. The study established a solid foundation of cooperative cancer gene therapy, and has considerable significance in developing safe and/or effective anti-tumor medicine.The recombinant expression plasmid of pVVP3IL-18HN was constructed by inserting VP3 gene and promoter-phored(IRES) IL-18HN fused geneinto the downstream of promoter(CMV). After identification through enzyme cut, the above recombinant expression plasmid was transfected into human hepatoma carcinoma cells SMMC7221, then the recombinant was screened by using RT-PCR, western-blot and indirect immunofluorescence. The results showed that the apoptin gene and IL-18HN fused gene were expressed correctly in hepatoma carcinoma cells and HN anchored on cytomembrane, however, apoptin concentrated in nucleus.Although the future of anti-tumor gene theraphy is optimistic, the therapeutic effect in vivo is not so good as in vitro. The main obstacle to carry out cancer gene therapy is the restricted transfection efficiency. It's evident that gene therapy depends on the ability that theoretical genes were transfected to target cells and were expressed as effector molecules. Gene therapy demands a high transfection efficiency, because residual malignant cells will make tumor recur. In order to transfect foreign genes to host cells, two carrier system including virus vector and plasmid vector have been developed. Because of virus vectors'toxicity and immunogenicity, its use may activate oncogene or inactivate tumor-suppressing gene. On account of the above reason, non-virus vector, especially plasmid vectors were more favored. However, its transfection efficiency is inferior to virus vectors. The plasmid that can arrive cellular nucleus and can be expressed is only accounts for one percent. In order to make the goal to cure diseases fully come true, large plasmid is needed and the required dose should be at mildigram grade. So the technology to prepare large-scale plasmid is needed to be developed to meet the needs of gene therapy and clinical experiments and lay a basis for the batch production after its going on sale. The constructed recombinant anti-tumor DNA was amplified in JM109strain of Escherichia coli. After fermated in complex medium, the fermentation technology of pVVP3IL-18HN was established. The Bacterium were 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 Q Sepharose XL column, and crude product was successfully isolated by continuous salt gradient elution. Then the crude product was applied to Source15 Q column for further purification. 131mg plasmid was got from 1044mg bacterium. 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, purity(OD260/280). Residual impurities levels such as RNA, host protein, host genomic DNA in final product were also detected. The result is as follows, pH 7.2±0.2, supercoiled form percentage 87.5%, trasfection effiency 6.20×10~5(cfu/μg plasmid), OD 260/280 1.86. host protein, RNA and genomic DNA were undetectable. And the purified plasmid was stable and can bear a certain high temperature. 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.