The Preparation Of NR1/TFR Recombined Epitope Vaccine And The Expression In Eukaryotic Cell

Abstract:Objective:N-methyl-D-aspartate receptor (NR) participated in many important physiology and pathologic reactions extensively, such as stress,cerebral injury,drug addiction and pain. Some diseases correlative with NR could be treated by the selective intervention of NR activities. NR artificial antagonists or blockers which are synthetic small molecule reagents, often caused blurred vision, anxiety, hallucinations and other serious side effects. Furthermore, it is difficult to sovle the problems of ultra-early management in clinical. NR1 is an essential subunit of the gene expression of NR complex. Therefore, preparation NR1 of oral vaccine by predicting epitope of its monoclonal antibody could intervene the stress, cerebral injury, drug addiction and pain in ultra-early management, when NR1 antigen through blood-brain barrier carried by Transferrin receptor (TfR) antibody. Consequently, our studies were to construct the eukaryotic expression plasmid of NR1-TfR fusion protein and B-cell epitope vaccine by phage display epitope prediction of MAB363 and OX-26 of mouse. Methods:(1) To filter the B cell epitope of mouse monoclonal [OX-26] to transferrin receptor by random-phage displayed dodecapeptide library. (filtration of the epitope of mouse anti-NMDAR1 monoclonal antibody MAB363 had already completed in our prophase experiment in same process, it was DDWVISTQSLKS). (2) The antigenicity and the space structure of fusion protein -S were detected by computer. (3) The confluence peptide (Pro-S) were detected examined in function, which connected the B cell epitope of MAB363 and OX-26 in series with a linker sequence. (4) The eukaryotic expression plasmid were constructed by inserted the foreign genes of NR1-TfR DNA fragments into the pcDNA3.1(+) plasmid vector, and NR1/TFR recombined epitope vaccine vaccine were prepared by transform vec-s into attenuation Salmonella typhimurium SL7207 by electroporation. (5) To detect the vec-s 'eukaryotic expression by western blot technology. Results:(1) The ascendancy epitope sequences of OX-26 monoclonal antibody were definited, the amino acid sequence was GHIHSMRHHRPT. The composite polypeptide whose sequences included GHIHSMRHHRPT and DDWVISTQSLKS, could indicate the specific binding with first antibody MAB363 or OX-26 by western blot. It was showed that the pcDNA3.1-NR1/TFR recombined epitope vaccine plasmid was professional designed, and the expressed protein could be effective and specifical bind with corresponding monoclonal antibody. (2) The composite polypeptide were predicted and simulated the space conformation of recombined epitope vaccine [protein-S] by DNAMAN,DNA STAR-PROTEAN software and online prediction software SMART and PredictProtein, which connected the B cell ascendancy epitope of MAB363 and OX-26 in series with a linker sequence. Therefore, it could be predicted better antigenicity and stimulate immunoreaction and generate homologous antibody. (3) The vec-s (pcDNA3.1-NR1/TFR), the eukaryotic expression plasmid, were successfully constructed and accurately trasformed into SL7207. (4) The vec-s were successfully transfected into eucaryote cells HEK293, and their expression were detected in 48h after transfection. Conclusions:We successfully constructed eukaryotic expression plasmid pcDNA3.1-NR1/TFR, and initially validated that vec-s could express objective protein in eucaryote cells. And it could build a good foundation for our next research.