摘要
为研制不带筛选标记的HIV活载体疫苗,首先构建含有neo基因和lacZ基因双重筛选标记的pVI75转移质粒,并将HIV-Ⅰ中国主要流行株B’/C重组株CN54 gagpol基因置于pVI75的启动子pE/L下,构建重组质粒pVI75-Gagpol。重组质粒与痘苗病毒天坛株共转染鸡胚细胞。前三轮通过G418加压,噬斑纯化,得到既含目的基因又含筛选标记的蓝色重组痘苗病毒;后三轮在无G418选择压力下,筛选只含目的基因而缺失了筛选标记的白色重组痘苗病毒。结果表明筛选到了一株重组病毒,经PCR和Dot blot检测确认该株重组痘苗病毒的neo基因和lacZ基因已丢失;PCR鉴定表明目的基因已插入重组痘苗病毒中;抗体染色和Western blot结果证实该重组病毒能很好地表达目的蛋白。
To develop live-vectored vaccine of Human immunodeficiency virus Ⅰ (HIV-Ⅰ) without selectable marker, we first constructed a transfer plasmid pVI75 with selectable markers of neo and lacZ gene, and a recombinant plasmid pVI75-Gagpol containing the gagpol gene of Chinese predominant prevalent HIV-Ⅰ strain CN54, pE/L upstream as the promoter. CEF was transfected by the recombinant plasmid pVI75-Gagpol, one hour after being infected with Tiantan vaccinia virus. A recombinant vaccinia virus rVV-Gagpol without selectable marker was constructed through two homologous recombinations as following: first, through three cycles of plaque purification under G418 pressure, the blue recombinant virus including both gagpol gene and the selectable marker gene was acquired; then through three cycles of plaque purification without G418 pressure, the white recombinant virus with gagpol gene but without the selectable marker gene was acquired. Thus, a recombinant vaccinia virus was acquired. PCR and Dot blot assay showed that the recombinant rVV-Gagpol lost the neo gene and lacZ gene. Gagpol gene could be detected by PCR. Antibody staining and Western blot results indicated this recombinant vaccinia virus could successfully express HIV Gagpol protein.
出处
《中国病毒学》
CSCD
2003年第5期441-445,共5页
Virologica Sinica
基金
国家863高新技术发展项目(2001AA21503)
