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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 eukaryotic recombinant vector containing human α-synuclein (hα-syn) encoding gene and express it in COS-7 cells, so as to lay the foundation for the development of nucleic acid vaccine for Parkinson’s disease. Methods The full-length CDS fragment of hα-syn gene was amplified by PCR, including KpnⅠ, XbaⅠand Kozak sequences. The amplified product and the eukaryotic expression vector pVAX1 were double digested, purified, ligated and transformed into competent cells E. coli TOP10. The recombinant vector pVAX1-hαS1-140 containing the gene of interest was screened and expressed in COS-7 cells. The expression products were detected by RT-PCR and Western blot. Results The single and double enzyme digestion confirmed that the inserted gene fragment was the same in size and the sequence and orientation of gene sequencing confirmed that it was completely correct. The results of RT-PCR and Western blot showed that the recombinant plasmid pVAX1-hαS1-140 can express the target protein in COS-7 cells and has biological activity. Conclusion The nucleic acid vaccine pVAX1-hαS1-140 was successfully constructed and expressed in COS-7 cells, which laid a good foundation for the study of nucleic acid vaccine for Parkinson’s disease.