Construction Of The Lentiviral Vector System Expressing NR2B Small Interference RNA

Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. It consists of two components,sensory discrimination and affective motivation.The former coding stimulation on the properties, including stimulation on the nature,intensity and location,which encode pain stimulation disgust,anxiety,fear and other negative emotions.Clinical observations have showed that patients with chronic pain,such as advanced cancer pain suffer the vicious emotional experience,for example,anxiety,fear,loneliness and even world-weary and so physical and psychological harm to patients caused by more serious than the disease itself.therefore,mitigation and treatment of chronic pain patients with malignant emotional experience is important for improving the quality of life and the treatment of disease.Anterior cingulate cortex(ACC)is an important part of the limbic system, which has extensive connections with other cortical areas and subcortical nuclei and is involved in modulating many functions. In recent years, an increasing body of evidence in eletrolphysiology,functional imaging and behavioral study indicates that ACC is closely involved in processing of pain, especially in the affective dimension of pain. NMDA (N-methyl-D-aspartate) receptor is a ligand-gated ionotropic glutamate receptor which involved in learning,memory,transcription of pain signal and other physiological and pathological processes.According to the different composition,the receptor can be divided into four subunits,2A,2B,2C and 2D.In ACC neurons,2B subunit is the main component.So our object is to construct a lentiviral vector of RNA interference(RNAi)of rat NR2B genes in ACC neurons.Method1 Three RNAi target sequences were designed according to the mRNA sequence of rat NR2B gene. The complementary DNA contained both sense and antisense strands were designed and synthesized.2 Double-stranded oligonucleotides synthesized should be cloned into the plasmid expression system pFU-GW-iRNA. The positive clones were screened by PCR and the recombinant vector was confirmed by DNA sequencing 3 Filter out the best NR2B-siRNA sequenceResultsIt was confirmed by digestion and sequencing that lentiviral vector had the correct structure. The vectors expressing 3 target sequences can infect 293T cells and the third vector has the best interference efficiency.ConclusionThe lentiviraI vector system has been constructed successfully.