Construction And Transformation Of Expression Vectors Including Amyloid β Peptide Gene

Amyloidβpeptide(Aβ) is one of the key targets in treatment of Alzheimer's disease. Now the Aβas immunogen used in immunotherapy for Alzheimer's disease is mainly gained by chemical synthesis with high cost and weaker immunity.It cannot meet the demand of Aβfor research and medical treatment.Therefore it is an effective method to produce Aβwith high immunity in bulk by genetic engineering technology.In this paper,we constructed prokaryotic expression vector and eukaryotic expression vector with tandem Aβgene.Then the expression of Aβgene in E.coli was investigated and transgenic tobacco plants were obtained.It established the foundation for manufacture of Aβvaccine by genetic engineering technology.The two tandem Aβgene(2Aβ) were obtained with PCR primers designed according to the base sequence of human Aβgene and the theory of overlap PCR.Then the prokaryotic expression vector pGEX-6P-1-Aβwas built by cloning 2Aβgene into vector pGEX-6P-1 (GST).The vector pGEX-6P-1-Aβwas transformed into E.coli BL21 and induced to express. The result of Western botting using GST antibody showed that the fusion protein GST/Aβwas expressed in E.coli but the expression level was low and the proteins were degraded partly.To establish high efficient expression vector,the plant expression vector pBIDST of double CaMV35S and TEV ehnancer was constructed.The results of GUS histochemical staining of transgenic tobacco plants indicated that the expression of gus driven by d35S-tev was higher than by CaMV35S.To investigate the expression of 2Aβgene in plant and its expression difference driven by different promoter,the expression vectors pBI121-Aβand pBIDST-Aβwere established. Then the tobaccos were infected by Agrobacterium tumefaciens EHA105 and the resistant plants were gained.The results of PCR and RT-PCR showed that the 2Aβgene driven by different promoter was integrated into the genome of tobacco and transcribed in tobacco.