pcdna3.1()icp27重組hsv2dna疫苗的構(gòu)建及在真核細(xì)胞中的表達(dá)

1、華中科技大學(xué)碩士學(xué)位論文pcDNA3.1(+)/ICP27重組HSV-2DNA疫苗的構(gòu)建及在真核細(xì)胞中的表達(dá)姓名：梁寧申請(qǐng)學(xué)位級(jí)別：碩士專業(yè)：皮膚病與性病學(xué)指導(dǎo)教師：李慎秋2011-04-29華 中 科 技 大 學(xué) 碩 士 學(xué) 位 論 文 3Construction of pcDNA3.1 (+) / ICP27 recombinant DNA vaccine against HSV-2 and its expression in eu

2、karyotic cells ABSTRACT Objective: To construct a new DNA vaccine against Herpes Simplex Virus type II and identify its expression in eukaryotic cells for the following research of immunogenicity of the vaccine . Methods

3、: Culture and amplificate HSV-2 virus in cultured vero cell, isolate total RNA, amplificate UL54 gene(expressing protein ICP27) of full-length by RT-PCR, and then connect UL54 gene to the pMD19-T Simple Vector and transf

4、orm into E. coli DH5α, extract plasmids. Make UL54 gene and eukaryotic expression plasmid —pcDNA3.1 (+) containing cohesive ends by KpnI and XbaI Double restriction enzyme digestion. Construct recombinant plasmid —pcDNA3

5、.1 (+) / ICP27 (viz. HSV-2 DNA vaccine), and then identificate if success by double restriction enzyme digestion and sequencing. Transfect the recombinant plasmid into COS-7 cells by method of liposome-mediated transfect

6、ion, identificate the transcription of HSV-2 DNA vaccine in cos-7 cells by RT-PCR and expression by Western blotting. Results: We successfully constructed pcDNA3.1 (+) / ICP27 plasmid: a 5428bp band (the pCDNA3.1 (+) vec

7、tor) and a 1551bp (HSV-2 UL54 sequence) band were shown by double restriction enzyme digestion; the DNA sequencing of UL54 was compatible with UL54 sequence in Genbank .After transfection into COS-7 cell, a length of 18

8、4bp UL54-specific fragments was successfully detected by RT-PCR, approximately 53kDa ICP27 was detected by Western blotting. Conclusion: We successfully constructed a new type of DNA vaccine against herpes simplex virus