The Preparation Of NR1/TFR Chimeric Epitope Vaccine And The Study Of The Immunoreaction In SD Rats

Objective: N-Methyl-D-Asparate (NMDA) type of glutamate receptor(NMDAR) could play key physiological and pathological roles in the central nervoussystem(CNS), widely participate in neural development, synaptic plasticity, learningand memory, ischemic brain injury and epilepsy and so on. NR’s antagonists orblockers of known now are synthetic small-molecule drugs and easy to pass theblood-brain barrier(BBB), but difficult to ultra-early administrate. Because of lowselectivity of drug action and often causing serious side effects such as confusion,anxiety, hallucinations, so they were difficult to use in clinical. NR1, basic subnits ofNR, have all electrophysiological and pharmacological properties of NR. By thetransferring receptor (TfR), NR1antibody maybe transited the BBB and played aninterventional role in the CNS. Therefore, NR1-TfR chimeric epitope oral vaccinecould build one of the feasible ways to ultra-early intervene to body stress, brain injury,pain, etc. In this study, we were to prepare of NR1-TfR chimeric epitope oral vaccinewhich had constructed the eukaryotic expression plasmid of NR1-TfR fusion proteinin the previous experiments, and could study the oral vaccines’ immunoreaction innormal SD rats. Methods:1)Eukaryotic expression plasmidpcDNA3.1-NR1/TfR(named for the vector-S) were expressed correctly in eukaryoticcells: the vector-S which had been constructed successfully in previous experimentswould be transfected into HEK293cells, and tested by immunoflurescence andwestern-blot;2) the preparation of NR1/TFR fusion epitope vaccine: The vector-S was electrotransformed into the attenuated Salmonella typhimurium (SL7207), it could betested by gene sequencing and filtered by Amp resistance, then the correct colonieswere cultured and named for SL7207-S. The oral vaccine (containing0.5X109cfu/mland1X109cfu/ml) would be preparated by plate culture counting method in OD600≈0.8.3) The study of the oral vaccines immunoreaction effects in SD rats: Thepreparated bacterin was fed by gavage n SD rats in four times within two weeks.During this period and after one month, observed all animals’ growth state, diarrhea ornot and death phenomenon, the intestinal mucosal immne in jejunum would beauthenticated by the immunohistochemical analysis, and the aimed antibody in serumand cerebrospinal fluid would be detected by immunofluorescence analysis withvector-S protein expressed in HEK293stabled as a known antigen. Results:(1) Takenthe commercial antibody MAB363of NR1and OX26of TfR as the primary antibodyto react with HEK293trancfected by vector-S, the red fluorescence was showed influorescence microscopy. The protein target band which were extracted from cells wasdetected by western-blot analysis within the marker range2KD to8KD.(2) Theplasmid S extracted from SL7207was comfirmed the same gene sequence to vector-S,SL7207-S bacterial content was1.6X107cfu/ml in OD600≈0.8.(3) Rats were noabnormal growth, no diarrhea or no death in all groups.(4) The brown substance wasdiscovered in rats’jejunum intestinal mucosal cells in the group2（oral1X109cfu/ml）by immunohistochemistry, but not in the blank group and group1(0.5X107cfu/ml).(5)The red fluorescence expressed in HEK293and did not exist in the blank group whenthe serum were regard as the primary antibodies in immunofluorescence, but it obviously expressed only in group2when the primary antibodies was cerebrospinalfluid.(6) After a month, the red fluorescence was still existed in serum in the groups,but brown substance and the red fluorescence in cerebrospinal fluid only in group2bythe same method. Conclusion:(1)The analysis of immunofluorescence andimmunohistochemistry showed that vector-S can be expressed correctly in eukaryoticcells afterHEK293are transfected, it was provided a basis for the next animalexperiments.(2) We were successful to obtain the SL7207-S bacteria and preparatedthe two concentrations of required oral vaccines.(3)The normal growth of groupsafter gavage with oral vaccine showed that the vaccine could be safely in oral andhave not remarkable bacterial toxicity.(4)The recombinant protein expressed byvector-S as the antigen was uptaked by the intestinal mucosal immune system andsuccessfully induced it.(5) Two concentrations of vaccine could induce the formationof circulating antibodies, and the antibodies which were induced by the oral vaccineconcentration of1x109cfu/ml could reachthrough the BBB into the cerebrospinal fluid.(6)The antibodies could persist in intestinal mucosal cells and cerebrospinal fluidbeyond a month.