Construction Of Targeted Composite Nogo Nucleic Acid Vaccine And Preliminary Evaluation Of Its Immunological Effects

Spinal cord injury(SCI)and other central nervous system injuries often result in paralysis.SCI can impose enormous pressure impact on individuals,families even the whole society.Studies have demonstrated that spinal cord injury regenerates difficultly compared with peripheral nervous system damage in the adult.There are many factors with effects on the repair and regeneration of CNS injury,among them the axonal growth inhibitory factor and the glial scar formation may be the most two important factors in the CNS microenvironment.Therefore,treatment through direct intervene inhibitory factor to break the signaling pathways and promote nerve regeneration,may be one of the effective therapy for spinal cord injury.As an important inhibitor,Nogo-A can inhibit axon regeneration and induce growth cone collapse through two targets of Nogo-66 and amino-Nogo,which mediat a variety of receptor inhibition signal of nerve regeneration.Nucleic acid vaccine,developed in recent years,is an effective strategy for the treatment of CNS injury,with the advantage of no harmful autoimmune response,no blood-brain barrier and micro-osmotic pump damage.Fms-like tyrosine kinase receptor-3 Ligand(Flt3L),an early hematopoietic growth factor,can be used as an immune adjuvant or tolerate stimulants to enhance the immune response.Then the targeted composite Nogo nucleic acid vaccine with FltsL,Amino-Nogo and Nogo-66 co-expression eukaryotic plasmid was constructed by genetic engineering methods.The animals were vaccinated and the antibody titer of anti-serum was detected.In order to observe the effect of vaccination on the spinal cord injured animals,the effects of antiserum on neurite outgrowth and neuroprotection were observed in vitro,the effects of the repair of spinal cord injury were observed in vivo,and the mechanism was further explored.The present study provids a novel method and theory for the treatment and pharmaprojects of the injured spinal cord and other central nervous system.The main methods and techniques1.Construction,detection and expression of the vectorThe target genes fragment Flt3L,Amino-Nogo,and Nogo-66 were amplified by polymerase chain reaction(PCR),then cloned into the plasmid pcDNA3.1(+)to construct the eukaryotic expression vector pcDNA-Flt3L-NogoN-Nogo66.The recombinant plasmids were amplified by transformation in E.coli,and were identified with the technology of PCR,restriction enzyme cut and sequencing separately.Then the recombinant plasmids were transfected into Chinese hamster ovary(CHO)cells through liposome,and the expression was analyzed by using immunofluorescence and Western-blot.2.Preparation and immune of targeted composite Nogo nucleic acid vaccineThe recombinant plasmid of pcDNA-Flt3L-NogoN-Nogo66 was amplified through transformation into E.coli DH5a.To obtain a large number of recombinant plasmid whitout toxins,endo-free plasmid maxi kit was used.The composite DNA vaccine was obtained by the recombinant plasmid conjugated with liposome as immune adjuvant with different ratio(2:1、1:1、1:2).Sprague Dawley(SD)rats were immunized once weekly for 6 weeks with 100 μg of the recombinant plasmid by injection into musculus tibialis bilaterally.In order to obtain the optimal praeparatum,anti-serum was isolated in blood taken from caudal vein,and the antibody titer of anti-serum was detected by enzyme-linked immunosorbent assay(ELISA).3.Observation of the effects of antiserum on neurite outgrowth and neuroprotection in vitro.As a neuron model,PC 12 cells were stimulated for 24 hours with anti-serum obtained by immunizing with above optimum formulations,then neurite outgrowth was observed directly by microscope,and apoptosis was detected by using flow cytometry to observe the neuroprotection effects.4.The immunological evaluation of targeted composite Nogo nucleic acid vaccineHalf-transection injury model of T9-T11 spinal cord was established in SD rats after 6 weeks immunized with complex Nogo DNA vaccine.To explore the immunological effects,the function recovery and axons regeneration in the spinal cord injured rats were evaluated through neurological behavioral,pathology-staining,nerve retro-tracer and immunohistochemistry methods.5 Preliminary safety testThe security of the DNA vaccine was preliminarily evaluated by observing the experimnetal allergic encephalomyelitis(EAE)and the change of weight during immune process in immunized rats.A small amount of heart,liver,spleen,lung,kidney,blood and muscle of injection site were collected,and then these tissues were amplified by PCR to observe the residual plasmid in vivo.The main results and conclusions1.The result of positive clone PCR,restriction enzyme cut and sequencing showed that the eukaryotic expression vector pcDNA-Flt3L-NogoN-Nogo66 was successfully constructed.The result of immunofluorescence and Western blot assay showed that the recombinant plasmid can successfully expressed of the corresponding fusion protein in vitro.2.The Nogo DNA vaccine which used liposome as adjuvant was successfully prepared.The corresponding anti-serum was obtained when animals immunized with the Nogo DNA vaccine.3.The result of anti-serum antibody titer test showed that the plasmid with liposome at the ratio of 1:1 is optimal praeparatum,and the immunogenicity of DNA vaccine is stronger to animals at this ratio.4.The antibody titer testing and in vitro experiments showed that the vaccine successfully stimulate the body to produce the corresponding antibody.The antiserum not only has a role in promoting the growth of axons of neurons,but also protects neuronal cell by inhibiting apoptosis.5.Half-transection injury model of spinal cord was successfully established.The immunological evaluation showed that the DNA vaccine can promote the recovery of motor function and spinal cord injury repair effectively.The DNA vaccine can inhibit Nogo signals which mediated inhibitory effect of nerve regeneration,and contribute to the improvement of motor function and spinal cord injury repair according to the results of neurological behavioral,pathology-staining,nerve retro-tracer,and immunohistochemistry.6.The weight,activity and diet in the immunated rats have no significant difference compared with those in the normal rats,and the results of PCR from different tissues were negative 6 weeks after spinal cord injury.These results showed that the targeted composite Nogo nucleic acid vaccine is relatively safe,and have no residue in vivo 6 weeks after injury.