hα-syn蛋白核酸疫苗pVAX1-hαS_(1-140)的构建及其表达

Construction and expression of nucleotide vaccine pVAX1-hαS_(1-140) coding human α-synuclein

目的构建含人α-突触核蛋白(humanα-synuclein,hα-syn)编码基因的真核重组载体,并在COS-7细胞中表达,为帕金森病的核酸疫苗开发奠定基础。方法通过PCR方法扩增hα-syn基因的CDS全长片段,含酶切位点KpnⅠ、XbaⅠ和Kozak序列。扩增产物和真核表达载体pVAX1经双酶切、纯化、连接,转化感受态细胞E.coli TOP10,筛选含目的基因的重组载体pVAX1-hαS1-140,并在COS-7细胞中表达,用RT-PCR法和Western blot法检测其表达产物。结果经单、双酶切证实插入的基因片段大小一致,基因测序证实其序列和方向完全正确。用RT-PCR法和Western blot法检测表明,重组质粒pVAX1-hαS1-140在COS-7细胞中能表达目的蛋白,而且具有生物活性。结论成功构建了核酸疫苗pVAX1-hαS1-140,并在COS-7细胞中表达,为帕金森病的核酸疫苗治疗研究奠定了良好的基础。

Objective To construct an eukarotic expression vector containing humana-synuclein（ha-syn） and be expressed in COS-7 cell,and lay the foundation for the exploiting nucleotide vaccine of Parkinson＇s disease（PD）. Methods The full-length CDS of has gene from were amplified using PCR,which containing restriction sites for the enzymes Kpn I and Xba I and Kozak consen- sus sequence. Then the amplificon and a eukaryotic expression vector pVAX1 were digested with Kpn I , Xba I simultanously,and were extracted and ligated by T4 ligase. The recombinant constructs pVAXI-hαS1-140 were transformed into competent E. coil TOP10 cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in COS-7 cells using RT-PCR and western blotting. Results The pVAX1 vector was successfully cloned with the has gene in the correct orientation and in-frame. The DNA vaccine constructs pVAXl-haS1-140with the hα-syn gene were shown to be expressed in COS-7. The hαS protein was successfully expressed in the mammalian cell line and was detected using RT-PCR and western blotting. Conclusion The promising results obtained in the present study have prompted further testing to improve the expression,and lay the foundation for the treatment of PD.