摘要
为构建O型口蹄疫病毒(FMDV)蛋白酶3C基因的真核表达质粒,并在BHK-21细胞中进行表达,以RT-PCR扩增O型口蹄疫病毒蛋白酶3C基因,利用XhoⅠ和HindⅢ限制性内切酶将3C基因克隆至真核表达载体pcDNA3.1(-),通过脂质体法转染重组质粒pcDNA3.1(-)-3C。PCR,双酶切鉴定及测序结果表明:重组质粒pcDNA3.1(-)-3C构建成功;Western-blot分析结果表明:重组质粒pcDNA3.1(-)-3C在BHK-21细胞中能够表达O型FMDV蛋白酶3C基因。该真核表达质粒的构建,为进一步研究O型口蹄疫空衣壳的体外组装以及O型口蹄疫空衣壳疫苗的开发建立了实验基础。
To construct the eukaryotic expression plasmid of tpye O foot-and-mouth disaese virus（FMDV） 3C proteinase, and to express in BHK-21 cells, 3C gene was amplified by reverse transcription-polymerase chain reaction （RT-PCR） from type O FMDV strain and then cloned into the eukaryotic expression vector pcDNA3.1 （-） using the restriction enzyme Xbo Ⅰ and Hind Ⅲ, the recombinant plasmid pcDNA3.1（-）-3C was transfected into the BHK-21 cells by Lipofectamine TM 2000 Reagent. After the confirmation by PCR, enzyme digestion and sequence analyses, the resulting plasmid pcDNA3.1（-）-3C was constructed correctly; Westernblot analyses showed that the plasmid pcDNA3.1（-）-3C was successfully expressed type O FMDV 3C proteinase in BHK-21 cells. The construction of the eukaryotic expression plasmid would help to study the assembly of FMDV empty capsid in vitro and establish a basic experiment to develop the type O FMDV empty capsid vaccine in future.
出处
《中国农学通报》
CSCD
2014年第20期17-20,共4页
Chinese Agricultural Science Bulletin
基金
中央级公益性科研院所基本科研业务费专项"动物病毒诊断新技术研究"(1610322013023)
关键词
O型口蹄疫病毒
3C基因
表达
foot-and-mouth disease virus type O
3C proteinase
expression
