Construction And Indentification Of The Recombinant Plasmid Co-expressing Aβ_3-10 Gene Of Alzheimer And CpG Gene

Construction and Indentification of the Recombinant Plasmid Co-expressing Aβ_3-10 Gene of Alzheimer and CpG GeneObjectiveWith the aging of population, more and more attention is paid to Alzheimer disease(AD), a representative disease of senile dementia. About 5 to 10 percent of people of sixty-five plus suffered from AD, the highest among senile dementia. It is investigated that there has been up to 120 million old people of sixty-plus in our country and 0.5 to 5.6 percent of them have dementia, in which AD accounting for fifty percent. So it is very important to explore the pathogenesis, diagnosis and prophylaxis of AD. Alzheimer disease(AD) is a primary neurodegenerative disease in central nerve system in middle or old people. It is characterized neuropathologically byβ-amyliod deposits, neurofibrillary tangles. In clinic, depressed ability of cognition and behavior disorder are often seen. The research results showed many reasons can lead to AD, but it is main cause thatβ-amyloid protein deposit, aggregate and forms amyloid plague. Although neurodegenetative diseases such as Alzheimer's are not classically considered mediated by inflammation or the immune system, in some instances the immune system may play an important role in the degenerative process. In 1999, the first immunotherapy for AD was performed with inoculating Aβ_1-42 on PDAPP mice. The experimental results showed Aβ_1-42 vaccine not only produced specific antiAβ_1-42-antibody, prevented and reduced Aβdeposition, but also improved cognitive behavior of PDAPP mice. But in the phaseⅡtrial, some AD patients(5%) who immunized with Aβ_1-42 suffered from intracranial infection and the trial had to been halted. A recent postmortem case report has shown some macrophages and T cell infiltrates in the CNS. It is possible that AD patients with high AβT cell reactivity deoelop severe T cell reactions in the CNS, when immunized and boosted Aβ. Nonetheless, titers of Aβantibodies did not correlate with the occurrence of severity of symptoms or relapses. So how to decrease T cell reaction immunized with Aβvaccine is effective messure to reduce side effects. Immunal therapy of AD is still attractive to the world even after several defeats. The future of immunal therapy becomes hopeful again by successive efforts of the reserchers.β-amyloid is a predominant component of senile plaques, one central pathological hallmark of Alzheimer's disease. Immunization with Aβcauses a marked reduction of Aβburden and its associated pathologies in brain. A clearance of the preexisting plaques has been observed in transgenic mice, with an inhibition of lesion of cognitive functions. No signs of an immune-mediated response could be detected to endogenous Aβpeptide of mice. The chronic inflammatory response resulted form immunization itself has not obviously impaired functions of central nerve system. Now reserchers pay attention to the application of micromolecularβ—amyloid peptide and N-terminal epitope, which is supposed to remove the plaques effectively and avoid inflammation reactions. The recent research have discovered that the non-methylized CpG motif which is ubiquitous in the bacterial DNA have a strong immunostimulating effect. The CpG ODN denotes the oligodeoxyribonucleotide which contains the cytosine-phosphoric acid-guanine motif(CpG DNA). The artifial oligodeoxyribonucleotide which contains the CpG motif(CpG ODN) can mimic the immunostimulating effect of the CpG motif in the bacterial DNA, and can induce the Th1-dominating immunoresponsibility. The combination of the CpG ODN and antigen of the protein-class can effectively induce the immunoresponsibility of the body, in which the CpG ODN have the effect of immunological adjuvant. Because of the poor immunogenicity of DNA vaccine we used the CpG-DNA as adjuvant to immoprove its immunogenicity. In order to search a more secure, efficient and cheaper treating project, we constructed plasmids which express Aβ_3-10 found in Alzheimer disease and CpG gene and investigate the possibility to make specific vaccine for Alzheimer disease with it. The results will provide foundation for further exploring the mechanisms of the elimination of the SP by active immunization to AB and developing DNA vaccine.MethodsAβ_3-10 cDNA was found in Genbank. Aβ_3-10 and Aβ_3-10-CpG gene segments which contain HindⅢenzyme site at the upstream and Eco RI enzyme site at the downstream were synthesized and then digested with HindⅢand EcoRI Aβ_3-10 and Aβ_3-10-CpG target gene segments were transferred into eukaryotic expression vector, pcDNA3.1(+). After Aβ_3-10 and Aβ_3-10-CpG eukaryotic expression vector was constructed, competent bacteria—DH5αwas converted. Plasmids were extracted. PCR was processed with pcDNA3.1(+) universal primer. Positive clone was picked out to identify recon with enzyme digestion. Sequencing proved its conformity with NCBI BLAST target gene, that indicated the vector was constructed successfully. Plasmid was extracted in small amount and transfected into Cos-7 cell line with liposome agent (Lipofectamine 2000) in vitro. Expression of pcDNA3.1(+)-Aβ_3-10 and pcDNA3, 1(+)-Aβ_3-10-CpG in eukaryotic cell was verified by Western-blot assay.ResultsEukaryotic expression vector of pcDNA3.1(+)-Aβ_3-10 and pcDNA3.1(+)-Aβ_3-10-CpG was constructed and verified by sequencing. The target segments were proved correct by comparison with NCBI BLAST soft ware. Expression of Aβ_3-10 and Aβ_3-10-CpG was detected in Cos-7 cell line which was transformed with the plasmid.ConclusionExpressing recombinant plasmid pcDNA3. 1(+)-Aβ_3-10 and pcDNA3.1(+)-Aβ_3-10-CpG can basically meet the requirement of Alzheimer's disease specific DNA vaccine, which can provide experimental date for the immunological effect of this candidate geneic vaccine in transgenic mice. The results will provide foundation for further exploring the mechanisms of the elimination of the SP by active immunization to AB and developing DNA vaccine.