人Tau基因多表位肽段原核表达载体的构建与表达

Construction of a prokaryotic expression vector of human tau multi-epitope peptide and immunogenicity of the expressed product

目的构建含人Tau多表位肽段基因的原核表达载体,在大肠杆菌中诱导表达并纯化目的蛋白,检测Tau多表位DNA疫苗免疫小鼠后特异性抗体产生情况。方法以质粒pVAX1-Tau为模板,用PCR扩增人Tau多表位肽段基因,插入表达载体pGEX-4T-2中,构建原核表达质粒pGEX-4T-2-TauP1/P2。将鉴定后的阳性重组质粒转入E.coli BL21(DE3)中,经异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,并采用GST法纯化、SDS-PAGE鉴定目的蛋白的表达。以TauP1/P2 DNA疫苗免疫小鼠获取血清,Dot-blot检测TauP1/P2特异性抗体产生情况。结果 PCR扩增出约300 bp的基因片段,克隆至表达载体后,经酶切和测序验证正确;获得了纯化的目的蛋白,并证实了GST-TauP1/P2融合蛋白的表达。Dot-blot检测到特异性TauP1/P2抗体的产生。结论成功构建和表达人Tau多表位肽段基因的目的蛋白GST-TauP1/P2,能够识别Tau多表位DNA疫苗免疫后小鼠产生的特异性TauP1/P2抗体。

Objective To construct a prokaryotic expression vector of human tau multiepitope peptide for examining the immunogenicity of a TauP1/P2 DNA vaccine in mice using the expressed product.Methods The coding sequence of Tau multiepitope peptide gene was amplified from the plasmid pVAX1-Tau by PCR and inserted into the prokaryotic expression vector pGEX-4T-2 to construct the recombinant plasmid pGEX-4T-2-TauP1/P2.The positive recombinants were transformed into E.coli BL21 cells,and the expression of fusion protein GST-TauP1/P2 was induced by IPTG and identified by SDS-PAGE. Mice was immunized with TauP1/P2 DNA vaccine and the production of the specific antibodies was detected by Dot-blot analysis using the purified fusion protein.Results A gene fragment 300 bp in length was amplified.Enzyme digestion and DNA sequencing verified correct construction of the prokaryotic expression plasmid pGEX-4T-2-TauP1/P2.The expression of target fusion protein GST-TauP1/P2 was detected by SDS-PAGE.Specific antibodies against TauP1/P2 were detected in the serum of mice immunized with the DNA vaccine using GST-TauP1/P2 fusion protein.Conclusion The constructed prokaryotic expression plasmid of human Tau multiepitope peptide is capable of expressing the target fusion protein,which specifically recognizes the specific antibodies against TauP1/P2 in mice immunized with TauP1/P2 DNA vaccine.