Preparation Double Targeted GMCSF-NgR-PirB For Nucleic Acid Vaccine And Its Immunotherapy For Spinal Cord Injury

Spinal cord injury is one of severe traumatic injuries in nerve canal neurons in perceptual and motor function of temporary or permanent defect,Because of the adult mammalian Central nervous system damaged its axon growth is restrained and affect the neural function regeneration and repair,the peripheral nervous system damaged nerve fibers were able to repair and restore its function.However,there are many influence factors in CNS injury repair and regeneration,On the one hand,because of nerve fiber limited ability to grow,the formation of glial scar and lack of neurotrophic factor of a variety of factors causing injury axon regeneration difficulty;On the other hand is due to the damage of central microenvironment of Myelin associated inhibitory factor and its receptor of existence,and thus mediated Myelin inhibit signal transmission.Therefore,through direct intervention of the central microenvironment inhibit signal transfer to promote the nerve regeneration,maybe as one of effective for spinal cord injury.As a common receptor of MAIs Nogo receptors and paired immunoglobulin-like receptor B mediated myelin inhibit transmission of signal with MAIs,may promote neural axon regeneration and protection of neuron growth cone,and reproductive inhibition effect into full play.Nucleic acid vaccine as a new immune treatment strategy in recent years has been widely applied to study the treatment of various diseases.Human Granulocyte macrophage colony factor can strengthen the body's immune response and the ability to promote T cell proliferation,is an important cytokine adjuvant,At the same time,GMCSF also has the nerve protective effect,can activate the nerve stem and progenitor cells and inhibit the formation of glial scar.To explore GMCSF-Ng R-Pir B mucleic acid vaccine immune therapy effect of damage of spinal cord injury,this research adopt the genetic engineering methods were cloned Ng R,Pir B and GMCSF gene fragments,build GMCSF-Ng R-Pir B eukaryotic expression vector with liposome form a composite mucleic acid vaccine immunization in rats.By using the methods of immunology,behavior and histology in vitro observation GMCSF-Ng R-Pir B mucleic acid vaccine helps central nevous axon regeneration and animal movement function recovery,to provide practical basis for study of spinal cord injury of immunotherapy.Main research contents:1.Construction,identification and its expression detection of vector co-expression GM-CSF,Ng R and Pir B gene fragments by PCR,were subcloned into pc DNA3.1(+)plasmid to construct recombinanted eukaryotic expression vector pc DNA-GMCSF-Ng R-Pir B by PCR,enzyme digestion and gene sequencing.Then,recombinant plasmid by lipofectamine transfect into CHO cells and immunofluorescence detect the protein expression of Ng R and Pir B.2.Preparation of double targeting GMCSF-Ng R-Pir B nucleic acid vaccineFirstiy,the liposome and GMCSF-Ng R-Pir B recombinanted plasmid were formed by the volume ratio of 1:1 to prepare the nucleic acid vaccine.Secondly,last six weeks,the SD rats were injected the nucleic acid vaccine in the muscle.The antibody levels were detected every week on each group.During immunization,the weight and behavior of animals were observed,and whether the vaccine can cause the occurrence of allergic encephalomyelitis in rats was also evaluated.3.Immunotherapy of double targeting GMCSF-Ng R-Pir B nucleic acid vaccine on the injured spinal cordAfter immunization,hemisection injury model in the left side of the spinal cord in rats was prepared,and the promoting effects of nucleic acid vaccine GMCSF-Ng R-Pir B on axonal regeneration and functional recovery were evaluated by behavioral,organization and molecular level.4.Detection of residued exogenous geneAfter six weeks,rats were immunized using the vaccine of GMCSF-Ng R-Pir B,and each tissue was extracted DNA as PCR template,to detect the target fragment of recombinant plasmid.Main conclusions:1.Co-expression vector of the eukaryotic expression for GM-CSF,Ng R and Pir B were successfully constructed.2.Nucleic acid vaccine of the double targeting GMCSF-Ng R-Pir B and the spinal cord injury of left side on SD rat model were prepared.3.Double targeting nucleic acid vaccine of GMCSF-Ng R-Pir B can stimulate the immune system to produce specific antibody,neutralizating of endogenous Ng R and Pir B,which effectivly reverse Ng R and Pir B in central nervous system which mediate signal transmission of myelin regeneration inhibitory,and promote the improvement of damaged.spinal nerve fiber and movement.