Construction Of The Expression Plasmid For NR1 Antigen And TFR Fusion Protein

Objective:N-methyl-D-aspartate receptor (NR) plays important roles in various physiological functions, such as synaptic plasticity, synaptic transmission, synapse formation underlying memory and learning during development. They are also associated with various pathological states including stress, drug addiction, pain, neurological disorders and psychiatric disorders. Excitotoxicity caused by overactivation of NR was a common pathological condition to these diseases. Selective intervention of NR activities could provide effective treatment to related diseases. The NR antagonists or blockers not only often caused blurred vision, anxiety, hallucinations and other serious side effects, but also there were some difficult problems to ultra-early management, poor access to clinical. NR1 is an essential subunit of the gene expression of NR complex, therefore, preparation NR1 oral vaccine by predicted epitope of it could solve the early intervention problems when NR1 antigen through blood-brain barrier carried by Transferrin receptor (TfR) antibody. So, our studies were to construct the expression plasmid of NR1-TfR fusion protein by phage display epitope prediction NR1 of mice, it was very important to preparation of NR1 oral vaccine. Methods:â‘ To determine the B cell epitope of a monoclonal antibody against NRl, a random-phage displayed dodecapeptide library was screened with the monoclonal antibody against NR1. After three rounds of biopanning, the peptide sequences of positive clones were determined and analyzed by DNA sequencing.â‘¡Coding Sequence (CDS) of mice was described in Genebank (NP₀35768). The gene (named as NRl-TfR) ecoding fusion expression of the mice TfR and NR1 epitope was made by polymerase chain reaction (PCR). Then, the plasmid of pUC57-NR1-TfR was completed.â‘¢NR1-TfR DNA fragments by double digested plasmid pUC57-NR1-TfR with KpnI and Xbal enzyme were cloned into the pcDNA3.1 plasmid vector. After translation Bacillus coli DH5a, the recombinant vector of NR1 epitope sequence and TfR gene fragment were successfully cloned and amplificated. The plasmid pcDNA3.1-NR1-TfR was evaluated by sequencing, electrophoresis after double digestion. Results:After 3 rounds of screening with the NR1 monoclonal antibody specifically binding phage were effectively enriched, DNA sequencing results showed that 54 monoclonal phage in all 56 sequencing expression for the same amino acid sequence:DDWVISTQSLKS, the other two single phage expression cloning of the amino acid sequences were:HSSHTSNTLPSV and TNTPPSQRVHLS. Digestion confirmed that mouse NR1 epitope sequence and sequence of mouse TfR gene fragment were successfully cloned into plasmid vector pcDNA3.1, the sequencing results that the recombinant vector of the design of the target gene sequence and fragments were exactly the same. Conclusions:Phage display random peptide library successfully screened with the NR1-specific antibody binding peptide DDWVISTQSLKS, the peptide analog could be a natural antigen epitope. There was a successful construction of the eukaryotic expression plasmid pcDNA3.1-NR1-TfR, which could express the fusion protein of the epitope of mice NR1 and TfR, it could build a test basis of oral vaccine for NR1.