摘要
目的构建HIV-1CN54株gagprotease基因嵌合脊髓灰质炎病毒cDNA的表达质粒,并鉴定、检测基因及其表达。方法用PCR技术获得人免疫缺陷病毒CN54株的gagprotease基因,并使其两端带上合适的酶切位点,将其定向插入到包含脊髓灰质炎病毒cDNA的表达质粒pSVA14中,替代其部分结构基因,构建HIV基因嵌合缺陷性脊髓灰质炎病毒基因组的表达质粒。经筛选、鉴定后用脂质体转染技术将新构建的质粒转入Hela细胞内,用WesternBlot方法检测目的基因在Hela细胞内的表达。结果PCR技术扩增所得的人免疫缺陷病毒CN54株gagprotease基因经琼脂糖凝胶电泳、DNA测序证实成功获得,未引入突变碱基,筛选、鉴定证明gagprotease基因被正确定向插入到脊髓灰质炎病毒的cDNA序列之中,WesternBlot检测到gagprotease基因正确表达了相关蛋白。结论成功构建了表达人免疫缺陷病毒CN54株gagprotease基因的缺陷性脊髓灰质炎病毒基因组嵌合质粒,为利用脊髓灰质炎病毒作人免疫缺陷病毒基因的表达载体奠定了基础,此研究对开发以脊髓灰质炎病毒为艾滋病的疫苗载体有重要意义。
[Objective] To construct a chimeric expression plasmid which contains HIV-1 CN54 strain gagpro- tease gene and Pohovirus cDNA, identify and examine the recombinant plasmid and its gene expression. [Methods] HIV-1 CN54 strain gagprotease gene with cleavage enzyme site in its two ends was obtained through PCR technique. It was orientedly inserted into the expression plasmid pSVA14, replacing one structural gene fragment of Polioviurs. Later the recombinant plasmid was proved correct construction by restriction enzyme cleavage identification. With liposome transfection way, the recombinant plasmid was trasnfected into cultured Hela ceils. Western blot was adopted to examine the gene expression. [Results] Proved through electrophoresis in gel, HIV-1 gagprotease gene was successfully amplified. No mutation occurred in its bases identified by gene sequencing. Identification by restriction endonuclease enzyme showed gagprotease gene was correctly inserted into poliovirus eDNA. Western blot test showed that HIV related protein was expressed in cultured Hela cells. [Conclusion] A chimeric Polioviurs-HIV gagproteae gene expression plasmid was constructed, which could provide the base for Poliovirus as a HIV gene expression vector. It is of great significance to invent an AIDS vaccine based on Poliovirus vector.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2006年第18期2721-2724,2728,共5页
China Journal of Modern Medicine
基金
This research was sponsored by National "863" Project Fund.
