摘要
目的构建天然溶瘤型新城疫病毒(NDV)Italien株血凝素-神经氨酸酶(HN)基因的真核表达载体并进行产物表达。方法采用RT-PCR法从病毒RNA中克隆HNcDNA,并构建HN的真核表达载体pcDNA3.1-HN,脂质体法转染CHO-K1,G418筛选稳定表达克隆。用Westernblot、免疫荧光法检测HN蛋白的表达。结果经限制性酶切鉴定及DNA测序分析,证实真核表达载体pcDNA3.1-HN构建正确。Westernblot和免疫荧光分析表明HN基因在CHO-K1中成功表达。结论天然溶瘤型NDVItalien株的HNcDNA被成功克隆,所构建的真核表达载体pcDNA3.1-HN可以在CHO-K1细胞中稳定表达。
Objective To construct an eukaryotic expression plasmid containing gene coding for the hemagglutinin-neurarninidase （HN） of newcastle disease virus （NDV） oncolytic strain Italien, and then to express the protein in eukaryotic cell. Method The HN cDNA was synthesized from viral RNA by RT-PCR, and the eukaryotic expression vector of HN gene （named pcDNA3. 1-HN） was constructed. The vector pcDNA3. 1-HN was transfected into CHO-K1 cell by liposome, and G418 was used to select stable clones expressing HN gene. The expression of HN protein was visualized by Western blot and Immunofluorescence microscopy. Results Restriction analysis and DNA sequencing proved that HN gene was correctly cloned into expression vector. Western blot analysis and immunofluorescence showed that the HN was expressed in CHO-K1 cells. Conclusion The HN cDNA of NDV was successfully cloned into eukaryotic vector which showed good expression of HN protein in CHO-K1 cells.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2007年第2期100-103,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助项目(30671196)
全军医药卫生科研项目(06MA218)
关键词
新城疫病毒
血凝素-神经氨酸酶基因
转染
基因表达
newcastle disease virus
hemagglutinin-neuraminidase gene
transfection
gene expression
