Effects Of RDP-p53Fusion Protein On IgG And Inflammatory Cytokine Of Mice

p53gene is an important tumor suppressor gene, its coded p53protein in cell cycle regulation, DNA repair and apoptosis induced has a key role, more than50%of human tumor tissues in p53gene mutation, is the most common is detected by the gene mutation, researchers have pointed out that wild-type p53protein function is the lack of necessary conditions of the tumor.P53protein into tumor cells can play a good antitumor effect, but the p53protein as a macromolecular material itself through the cell membrane into the tumor cells, make its application is limited by a certain.Kumar, the study found that comes from the rabies virus capsid glycoprotein of peptides derived from RDP (rabies virus get glycoprotein derived peptide) can carry antiviral siRNA through blood-brain barrier that helps to the mice infected with encephalitis, brain greatly improve the survival rate of encephalitis in mice, and RDP as carrier has higher security.Kumar, such as the pioneering study of RDP to solve large molecules is difficult to through the cell membrane and blood-brain barrier into the brain problem provides a good foundation of previous successful use of our laboratory RDP BDNF macromolecular protein specificity quickly into the brain.Ever wear usually by the cell membrane in the study of peptide (cell-penetrating peptides, CPP) carry large molecules into cells, RDP peptides as a new type of brain targeting deliver medication carrier overcomes the defect of the CPP no tissue specificity.This experiment using biotechnology by e. coli RDP-p53, fusion protein expression, in order to carry through the brain targeting drug carrier RDP p53across the blood-brain barrier, and to develop new drugs for the treatment of glioma brain tumors such as.Biotech drugs in the body is one of the most common problems of the immune response, it not only reduce the drug efficacy, and damage to the body, the immunogenicity of evaluation as to clarify the key factor for clinical safety and efficacy of these drugs, evaluating the expect is the immunogenicity of biotech drugs is an important content of preclinical and clinical evaluation.This experiment by ELISA to detect the levels of serum IgG to evaluate the strength of the immunogenicity.P53, besides have antitumor effect, also contact between certain inflammatory mediators, p53can affect inflammatory cytokines (inflammatory cytokine) secretion;Fusion protein as exogenous substances of the body, may activate the body’s own immune response, triggering autoimmune inflammation.Studies have shown that the body to release a series of inflammatory cytokines on the expression of drug metabolism enzymes and functional regulation, the body to a drug disposal process of significant change will happen.Therefore, the inflammatory cytokines may affect RDP-the pharmacological effects of p53fusion proteins.This experiment by ELISA to detect serum IL-1beta, IL-6, the level of TNF alpha to analyze intraperitoneal injection of different doses of RDP-p53protein in mice serum level of inflammatory cytokines for fusion research RDP-p53protein of laying a foundation for the pharmacological effects of glioma.The experiment was divided into two parts:The coding sequence of p53was amplified by PCR using plasmid pET28a-p53as a template and cloned into expression vector pET28a-RDP. The constructed recombinant plasmid pET28a-RDP-p53was transformed into E.coli Rosetta, protein expression was induced with IPTG and protein was purified. The expressed product was identified by SDS-PAGE.Second:Then Kunming mice were divided into4groups, i.e.blank control group (constant volume normal saline) and RDP-p53fusion protein high-dose, medium-dose and low-dose groups (4mg/kg,2mg/kg,lmg/kg). Kunming mice were immunized by peritoneal, once2d, of which the sera were separated30d later and determined for IgG and inflammatory cytokine (IL-1β, IL-6and TNF-α) level by ELISA.Results:Both restriction analysis and sequencing proved that p53gene was cloned into expression vector pET28a-RDP; RDP-p53fusion protein got expression in both supernatant and precipitation.ELISA results showed that:compared with control group, IgG content of low-dose and medium-dose groups didn’t increase significantly (P>0.05), IgG content of high-dose groups increased significantly (P<0.05).compared with control group, inflammatory cytokine content of low-dose and medium-dose groups didn’t increase significantly (P>0.05), IL-1β and TNF-α content of high-dose groups increased significantly (P<0.05)Conclusion:RDP-p53fusion protein was successfully expressed and purified, it had weak immunogenicity and the immunogenicity was related to dosage of administration,1mg/kg and2mg/kg doses in mice by intraperitoneal injection of repeated RDP-p53fusion protein will not prompt a significant rise in inflammatory cytokine levels, this might provide some valuable information for determining dosage of administration while studying pharmacological effect that RDP-p53fusion protein inhibits brain tumors.