Construction Of DNA Vaccines Containing Alpha-Synuclein And Their Neuroprotective Effects

BackgoundParkinson's disease(PD) is a progressive neurodegenerative disorder of the central nervous system.Latest studies have showen that there are more than 4 million individuals affected with PD worldwide currently and 1.7 million in China and 0.5 million in the United States.It is a huge financial and spirital burden for the families and society.In the 21st century Chinese population become aged with the most amount of old people in the world,China is facing the very serious and irreversible situation of population. The number of people over 60 years old is projected to increase more than threefold by 2050.The prevention and treatment of PD is an important public health problem.The most obvious brain structure affected by PD is a part of the substantia nigra pars compacta and apparent loss of dopaminergenic neurons pathologically.Aggregation ofα-synuclein is one of main pathological feature of PD. Lewy body in remaining neurons of the substantia nigra is the main pathological feature of PD in whichα-synuclein is the major constituent.So far the treatment of PD can not be against theα-synuclein. At present the drugs against PD only aim at the DA metabolism.Levodopa,the main drug,can not stop the progress of the disease, but also may result in a serious complication in 2~5 years.So scientists have been struggling to explore novel treatments.The treatments of PD include neurotrophic factors, transplantation of neural stem cell, antibodies, vaccines and traditional chinese medicine. Immunotherapy is an easy, economical and effective way.In immunotherapy,the application of vaccines is growing, not only for the prevention and treatment of infectious diseases, but also for non-infected diseases,cancers and autoimmune diseases. Vaccines against neurodegenerative diseases have become a focus.Many studies have been conducted on the vaccines of Alzheimer's disease.But the vaccine of PD is still in its infancy.Benner made the first exploration on the vaccine of PD,and he found that immunotherapy with copolymer-1 immune cells might reduce neuro- degeneration in the MPTP model.Masliah inoculated hα-syn transgenic mice with recombinant humanα-syn, and the vaccination led to a specific nervous protection. High affinity antibody was generated, and antibody identified epitopes within the C-terminus region of humanα-syn in the vaccinated mice; the toxicα-syn was cleared via lysosomal activation, aggregates ofα-syn were reduced in the DA cells and synapses. These findings suggest that immunization with associated antigen may be a breakthrough of PD.Lewy bodies contain many proteins,such asα-syn, ubiquitin,Parkin,14-3-3,synphilin-1 and Ubiquitin C-terminal hydrolase L1 (UCH-L1).Abnormalα-syn plays a key role in the pathogenesis of PD. Soα-syn protein may be a promising therapeutic target in PD.Studies have shown that active immunization may evoke the harmful inflammation which is associated with the activation of immune cells.The recombinant vaccine may also encounter this problem. However, the immunoadjuvants may bring a new hope for vaccines. Especially there is many immunoadjuvants for DNA vaccines. Appropriate adjuvants may resolve the problem. Immunoadjuvants may enhance the immunogenicity or change the type of immune response. The adjuvants of DNA vaccine are DNA fragments, such as cytokines, Cytosine Phosphate Guanidine(CpG)and costimulators. IL-10 is a important immunomodulatory cytokines which may provide a mechanism for evading the T cell-mediated immune response.So we fused IL-10 gene and humanα-syn gene together via genetic engineering techniques in this issue, constructed DNA vaccines pVAX1-hαS140 and pVAX1-hαS140-IL-10, immunized PD mice, and expected to induce the protection of nervous system and inhibit the inflammation.Objective : To amplifyα-syn gene from human embyo brain,and amplify IL-10 gene from human lymphocytes.To clone humanα-syn gene into pVAX1 to construct recombinant plasmid pVAX1-hαS140, and fuse humanα-syn gene and IL-10 gene to construct recombinant plasmid pVAX1-hαS140-IL-10.To observe the expression of recombinant plasmids in COS-7 cells and identify their biological activity.To vaccinate normal C57BL/6 mice and observe the effect of humoral immunity.To inoculate PD mice and observe neuroprotective effects of the DNA vaccines, and lay the foundation for the exploiting DNA vaccine against PD. Methods :1.Construction and expression of recombinant plamids pVAX1-hαS 140 and pVAX1-hαS140-IL-10.The hα-syn gene was amplified from embyo brain tissue using RT-PCR,which containing restriction sites for the enzymes KpnⅠand XbaⅠand Kozak sequence. The IL-10 gene was amplified from human limphocytes.The recombinant plamids pVAX1-hαS140-IL-10 and pVAX1- hαS140 were constructed.The recombinant constructs were transformed into competent E.coli DH5αcells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. Western blot was used to detect the expression of the constructs in COS-7 cells.2.Immunization of the DNA vaccines pVAX1-hαS140 and pVAX1- hαS140- IL-10.Recombinant ultrcoiled plasmids were purified with high yields. The normal C57BL/6 mice were immunized with pVAX1,pVAX1-hαS140 and pVAX1-hαS140-IL-10 respectively. ELISA was used to detect the titer ofα-syn antibodies. The binding of the mice sera to humanα-syn was analysed by immunohistochemistry and hα-syn neutralization test.3.Neuroprotection of the DNA vaccines pVAX1-hαS140 and pVAX1- hαS140-IL-10 against PDNormal C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine subcutaneously. These models were immunized with pVAX1-hαS140 and pVAX1-hαS140-IL-10.The tyrosine hydroxylase(TH)andα-syn were analyzed by immunohistochemistry to compare the neuro- protective effcets of of DNA vaccines.Results1. Construction and expression of recombinant plamids pVAX1-hαS 140 and pVAX1-hαS140-IL-10. The pVAX1 vector was successfully cloned with the hα-syn gene in the correct orientation and in-frame.The recombinant constructs pVAX1- hαS140 and pVAX1-hαS140-IL-10 were expressed in COS-7 cells.2.Immunization of recombinant plamids pVAX1-hαS140 and pVAX1- hαS140-IL-10Antibody titers in animals belonging to pVAX1-hαS140-IL-10 group were higer than that of the pVAX1-hαS140 group(P<0.01).The highest antibody titers were more than 1:5000.3.Neuroprotection of DNA vaccines pVAX1-hαS140 and pVAX1- hαS140-IL-10 against PDVaccinations of DNA vaccines cleared aggregates of overexpressedα-syn in the brain of PD mice partly.Fusion plasmid pVAX1-hαS140-IL-10 has a better immune effect than pVAX1-hαS140(0.01