Research On The Anti-tumor Activity Of Polyarginine-Cytosine Deaminase Fusion Protein

The tumor is a serious threat to public health, the leading cause of death worldwide.Although the research of the mechanism of cancer has made great progress, but there still a longway to go before thorough treatment of cancer.Biological macromolecules such as proteins play an important role in the occurrence andtreatment of many diseases. However, according to Lipinski’s rule of five, we usually thinkprotein drugs have low permeability. Hydrophilic molecules like proteins have therapeutic valuebut no cell membrane-permeable and easily biodegradable greatly restricted in the field of cellbiology and pharmacy. For a long time researchers committed to exploring these effectivemolecules into living cells. Common methods have physical methods such as electroporation,microinjection; biological and chemical methods, such as treated with the digitonin, thepore-forming protein; other methods such as the use of protein carrier immunotoxins andparticles such as liposome and the viral vector. Although these methods have their ownadvantages, but all have problems like low transduc efficiency, easily lead to cell damage andeven death, complex and difficult to regulatory and other issues.Cell penetrating peptides(CPPs), also known as protein transduction domain (PTD), Troypeptide. Typically comprising5-30amino acids, unlike most peptides, cell penetrating peptidescan non-cell-specific, non-receptor-mediated import freely into a variety of mammalian cells,while will not cause cell damage. The discovery of cell penetrating peptide brings the dawn ofbiological macromolecules for the treatment of disease. Cell penetrating peptides also has agood application prospect in the field of cell biology, transportion of the drug in body, clinicalefficacy evaluation and cellular immunology research. At present widely recognized simplestand the most effective cell penetrating peptide is polyarginine.Cytosine deaminase (CD) is a typical suicide gene for cancer therapy. Cytosine deaminasecan convert preamble non-cytotoxic prodrug5fluorocytosine (5-FC) to a chemotherapy drug5-fluorouracil (5-FU).5-fluorouracil can block DNA replication by inhibiting thymidylatesynthase activity in tumor, then induce apoptosis.The cytosine deaminase used in this study come from Escherichia coli, bacterial cytosinedeaminase and its mutant. This mutant replaced natural prokaryotic cytosine deaminase at site316and317phenylalanine and aspartic to cysteine and glycine. Mutated prokaryotic cytosine deaminase can significantly improve the efficiency of conversion of5-fluorocytosine, show astronger tumor suppressing ability.In this study, we builted a pSUMO-R9-EGFP vector on the backbone of original pSUMOvector, then transformed pSUMO-R9-EGFP into E.coli Rosetta (DE3). After IPTG inducted, theexpression and solubility of the SUMO-EGFP and SUMO-R9-EGFP were analyzed bySDS-PAGE. Ni-NTA affinity chromatography was used to purify SUMO-R9-EGFP and addedSUMO protease1to digest SUMO tag, and ultimately, obtained the mature R9-EGFP fusionprotein with high purity. The penetrating ability of the R9-EGFP on HepG2was determined byflow cytometry and confocal laser microscope. The results show that mature R9-EGFP canacross cell membrane into the cell interior, and gather at nucleus, while EGFP does not have thisability. The penetrating efficiency show time and dosage dependence, could be inhibited byheparin.Bacterial cytosine deaminase and its mutant were inserted into the EGFP site of the plasmidpSUMO-R9-EGFP, and successfully purified polyarginine-bacterial cytosine deaminase andpolyarginine-bacterial cytosine deaminase mutant. The cytotoxic effect of these two fusionproteins on HepG2and U251was determined by MTT assay. The results show that both thesetwo fusion proteins are cytotoxic, and the mutant is superior to the natural bacterial cytosinedeaminase, yet bacterial cytosine deaminase alone has no significant cytotoxicity. Significantdifference between the results.We also established the H22tumor model in Kunming mouse, polyarginine-bacterialcytosine deaminase was subcutaneous injection with abdominal injection of5fluorocytosine.Results show that these two fusion proteins have anti-tumor activity compared with theuntreated group and individual cytosine deaminase treatment group. The fusion proteintreatment groups have a prolonged survival time of8-10days.