The Construction And Preparation Of A DNA Vaccine Targeted NR2B Epitope And Its Analgesic Efficacy Experimental Study

BACKGROUNDPain is difficult to manage and has a variety of clinical symptoms. Peripheral nerve injury often results in neuropathic pain. Diabetes, infection (herpeszoster), nerve compression, nerve trauma,''channelopathies,''and autoimmune disease are examples of diseases that may cause peripheral nerve injury. Neuropathic pain characterised by hyperalgesia (increased pain elicited by noxious stimuli), allodynia (pain from normally innocuous stimuli) and ongoing pain are difficult to treat. The hyperalgesia and ongoing pain may correspond to peripheral sensitization and bursts of spontaneous activity in the injured afferents related to sodium channel dysfunction. The allodynia is due to central sensitization. The N-methyl-D-aspartate (NMDA) receptors mediate central sensitization and peripheral sensitization and visceral pain during pain states. Among NMDA receptor subtypes, the NR2B subunit containing receptors appear particularly important for nociception, thus leading to the possibility that NR2B selective antagonists may be useful in the treatment of chronic pain. However, several NR2B selective agents seem to block human ethera-go-go-related gene (HERG)-mediated K + currents and may thereby produce severe side effects by increasing the cardiac Q/T interval. DNA vaccinization induces immune responses by direct injection into animals of DNA encoding antigenic proteins. The use of molecular methods, such as gene therapy, stem cell therapy and viral vector for delivery of biologic antinociceptive molecules, has led to a better understanding of the underlying mechanisms of induction of intractable neuropathic pain.DNA vaccine consists of a vector plasmid and an antigen-encoding gene. The DNA plasmid is taken up by host cells where the encoded protein is made. The protein was presented by APC via MHC molecularⅠorⅡto elicit both humoral and cellular immune responses. DNA vaccine has proven to be effective in small animal models for viral, bacterial diseases, tumor, and autoimmune -diseases. In the present report, we focus on the construction and preparation of a DNA vaccine targeted NR2B epitope and its analgesic efficacy experimental study.METHODS and RESULTS1. To screen the mimic epitope of N-methyl-D-aspartate receptor 2B subunit from phage display peptide libraryMethods: The monoclonal antibody interacting with NR2B was used as target protein to screen the binding peptide from a 12-mer Ph.D random peptide library. After three rounds of affinity screening, twelve specific clones were selected randomly and identified by sandwich ELISA. The peptide sequences were analyzed by DNA sequencing. The clones containing a common sequence were named positive clone. The competitive inhibition of the native antigen bound to monoclonal antibody against the NR2B by the positive clone was assayed by cell ELISA.Results: After 3 rounds of screening the phages specifically bound with mAb NR2B were selected and amplified. Nine of the 12 clones displayed a common DNA sequence was 5′- TCG CAT CCG CCT GTG ATG CCT TGG CCT ACT TCG ACT-3′, aminoacid sequence: SHPPVMPWPTST. The inhibitory assay showed that the mimic epitope peptides displayed on the phage surface could effectively inhibit the combination of the monoclonal antibody with native antigen. The inhibitory rate of mimic epitope was (45.2±3.4) %. 2. Construction and identification of a DNA vaccine targeted NR2B epitopeMethods: The target fragment of NR2B epitope was obtained by PCR, then inserteded in the expression vector pDC515 by DNA recombinant technology and constructed pDC515/eNR2B. The recombinant plasmid pDC515/eNR2B was identified using PCR, restricted endonuclease and DNA sequence analysis. E.coli DH5αwas transformed with the pDC515/eNR2B and then large-scale prepared and purified the pDC515/eNR2B. HEK 293 cells were transfected with the recombine plasmid pDC515/eNR2B by 293fectinTM. RT-PCR and immuncytochemistry staining method were used to detect the integration and expression of NR2B epitope gene in transformed 293 cells.Results: The restriction endonuclease digestion and DNA sequencing confirmed that the recombinant plasmid pDC515/eNR2B containing a mimic epitope of NR2B. A DNA vaccine targeted NR2B epitope was large-scale prepared and purified by transformation E.coli DH5α. PCR and RT-PCR products were analyzed using 1.5% agarose gel electrophoresis, the size of amplification product of target gene was identical to the positive (80bp), but there was no PCR and RT-PCR product from non-transfected cells. HEK 293 cells transfected were stained positively.3. Studies on the immune profile of a DNA vaccine targeted NR2B epitopeMethods: 40 adult male Sprague- Dawley(SD) rats weighting 250g-300g were randomly divided into 4 groups,10 rats for each group:pDC515/eNR2B group, pDC515 group, PBS group and Na?ve group. 100μg pDC515/eNR2B or pDC515 or100μl PBS were injected into the quadriceps muscles of rats in pDC515/eNR2B group, pDC515 group and PBS group respectively at days 0, 7. The special antibody against NR2B of the rat serum was assayed by ELISA at days before immunization and 8d, 14d 21d after the second immunization. The combination activity of the special antibody against NR2B of the immunized rat serum was analyzed by immunohistochemical and Western-blot analysis. The effect of immunization with a DNA vaccine targeted NR2B epitope on the cultured PC12 cells proliferation was investigated with MTT. The rats'immunized serum on the intracellular Ga2+ ion in PC12 cells damaged by Glutamate was observed by flow cytometry.Results: The specific antibody against NR2B was induced to develop in the serum of rats immunized with pDC515/eNR2B at 8d after the second immunization and lasted in high titer to 21d. There was no specific antibody of NR2B in pDC515 group,PBS group and naive group(P <0.05). The specific antibody against NR2B in the serum of rats immunized with pDC515/eNR2B could combine with itself lumbar spinal cord. The concentration-dependent enhance effect on PC12 cell proliferation was observed by MTT in the serum of pDC515/eNR2B group, pDC515 group,PBS group and naive group(P >0.05), but the serum of rats immunized with pDC515/eNR2B could reduce the intracellular Ga2+ ion concentration in the PC12 cells damaged by Glutamate( P<0.01). However, this reduction was not detected in the serum of rats immunized with the pDC515 and PBS.4. Effect of vaccination with a DNA vaccine targeted NR2B epitope on chronic neuropathic pain in SD-ratsMethods: 80 adult male Sprague- Dawley(SD) rats weighting 250g-300g were randomly divided into 5 groups,Na?ve group(n=7), SNI group(n=42), Sham group(n=7), SNI+pDC515/eNR2B group(n=12), SNI+pDC515 group(n=12). Rats in the SNI, SNI+ pDC515/eNR2B and SNI+ pDC515 groups underwent left side spare nerve injury (SNI) operation. Rats in the SNI+ pDC515/eNR2B group, the SNI+ pDC515 group were injected into the quadriceps muscles twice with 100μg pDC515/eNR2B or pDC515 respectively at days 5 and 12 post SNI surgery. The SNI group (n=42) was devided into 4 subgroups: SNI group (n=8), SNI+Ifen group (n=12), SNI+Morph group (n=12), SNI+mAbNR2B group (n=10). Rats in the SNI+Ifen group and SNI+mAbNR2B group were injected into intraperitoneal twice Ifenprodil (a selective NR2B antagonist) and mAbNR2B respectively 10 mg/kg at days 21 and 26 post SNI surgery. Morphine was acute administered 15 min (10 mg/kg, i.p.) prior to behavioral testing. The withdraw threshold by mechanical touch allodynia were measured at days -1d,5d,12d,20d,25d,30d,37d post SNI surgery. The serum special NR2B antibody was determined by sandwich ELISA in the Na?ve group,SNI group(n=8),Sham group,SNI+pDC515/eNR2B group and SNI+pDC515group at days -1d,5d,12d,20d,25d,30d,37d post SNI operation. The NR2B expression of rats'spinal cord was determined by immunohistochemical analysis and Western-blot. The expression of astrocyte, microglial and cytokine IL-1βof spinal cord were detected by immunohistochemical analysis. The effect of the serum of rats immunized, Ifenprodil, mAbNR2B and Morphine on the intracellular Ga2+ ion in PC12 cells damaged by Glutamate was observed by flow cytometry.Results: All animals performed SNI surgery developed qualitative signs indicative of a marked sensory hypersensitivity of the hindpaw ipsilateral to the nerve injury at day 5 post surgery and the withdrawal duration apparent increase and withdrawal threshold significant reduction, compared with Na?ve group, P<0.05. Immunization with the DNA vaccine targeted NR2B epitope profoundly prevented the increasing in paw withdrawal duration to mechanical allodynia and the analgesic effect is similar to that given Ifenprodil (10 mg/kg, i.p.) and less than that achieved with acute administration of 10 mg/kg of Morphine, P<0.05. NR2B expression in lumbar spinal cord increased after SNI surgery and immunization with the DNA vaccine targeted NR2B epitope or treatment with mAbNR2B could reverse this change( P<0.05). The astrocyte and microglial were activated in SNI group and cytokine IL-1βexpression was increased( P<0.05). The activation of astrocyte and microglial were prevented in the SNI+Ifen group and SNI+Morph group( P<0.05). The activation of astrocyte and microglial and over expression of cytokine IL-1βof spinal cord could detection in SNI+pDC515/eNR2B group and SNI+mAbNR2B groip. Moreover, the immunization serum with the DNA vaccine targeted NR2B epitope and treatment with Ifenprodil or mAbNR2B reduced intracellular Ca2+ ion concentration in Glutamate damaged PC12 cells, but this reduction was not detected as acute administration Morphine.5. Statistical analysisData are presented as mean±standard deviation (SD), the withdraw threshold using a One-Way repeated measures ANOVA and the other results using one way analysis of variance (ANOVA) by SPSS 11.0. A value of P<0.05 was considered statistically significant.CONCLUSIONS and SUMMARYThe mimic epitope of NR2B was screened successfully from the 12-mer Ph D random peptide library. This peptide mimics an epitope of the native NR2B. A recombinant plasmid pDC515/eNR2B was successfully constructed and a DNA vaccine targeted NR2B was prepared and purified. This DNA vaccine targeted NR2B epitope could induce humoral immune response in SD rats to develop special antibody against NR2B.This DNA immunization and immune response have no effect on the PC12 cells proliferation. The special antibody against NR2B of serum immunized could prevent the increase of Ca2+ ion intracellular in PC12 cells damaged by Glutamate. Immunization with the DNA vaccine targeted NR2B epitope could alleviate allodynia and hyperalgesia behaviour after chronic neuropathic pain, which is associated with reverse NR2B up-regulated and then reduction intracellular Ca2+ ion concentration. The analgesic efficacy of DNA vaccine targeted NR2B epitope is similar to that given Ifenprodil or mAbNR2B and less than that achieved with acute administration of Morphine.To screen the mimic epitope of N-methyl-D-aspartate receptor 2B subunit from phage display peptide library and prepare a DNA vaccine targeted NR2B by gene recombinant technology. To achieve significant analgesia effect on rats neuropathic pain by DNA immunization. Therefore, DNA vaccine and the initial picture of their utility in neuropathic pain are emerging. However, further analysis is required to determine their ultimate efficacy and mechanisms.