摘要
根据GenBank中的猪细小病毒(PPV)VP2基因序列设计并合成了2对突变引物,对PPV SC-1株VP2基因进行了定点突变。利用重叠延伸PCR技术分别将VP2蛋白中第402位(A)和214位(B)的半胱氨酸突变为精氨酸,并定向克隆入真核表达载体pPI-2.EGFP中,构建含报告基因EGFP的重组质粒pPI-2.EGFP.VP2(A)、pPI2.EGFP.VP2(B);利用脂质体介导法将上述重组质粒转染COS-7细胞,从而研究该突变对VP2基因表达的影响。结果显示,成功构建了含突变体及EGFP的真核表达载体;在倒置荧光显微镜下,突变后的质粒转染呈现不同的荧光信号,A样品的荧光呈颗粒状分布于细胞中,而B样品与阳性对照相似,荧光信号均匀分布于细胞中。提示,突变的引入是导致表达差异的主要原因。
Based on the VP2 gene sequence of porcine parvovirus （PPV） available in GenBank, two pairs of primers were designed and synthesized to modify VP2 gene of the PPV SC 1 strain . Using overlap ping extension PCR,the codons encoding cysteine of VP2 protein in 214th and 402nd site were mutated into the codons encoding arginine, then the mutant VP2 gene was cloned into eukaryotic expression vector pPI-2. EGFP with green fluorescent protein（GFP） gene to construct the recombinant plasmids pPI-2. EG FP. VP2（A） and pPI-2. EGFP. VP2（B）. To elucidate the effect of the mutation on the expression of the mutants,the COS7 cells were transfected with the plasmids, via mediation by LIPOFECTION Reagent 2000. The result showed that the recombinant plasmids pPI-2. EGFP. VP2（A） and pPI-2. EGFP. VP2（B） were constructed successfully. The granular enhanced green fluorescent protein（EGFP） in the cells transfected by pPI-2. EGFP. VP2（A） was observed. This observation indicated that the mutants were introduced successfully into the target sites and the eukaryotic expression vectors containing both mutant and EGFP were constructed. The introduction of mutant was the main reason for the differential expression of plasmids.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第7期563-568,共6页
Chinese Veterinary Science
基金
国家自然科学基金项目(30500019)
四川省科技厅科技攻关计划项目(2004D004)
关键词
猪细小病毒
VP2基因
定点突变
真核表达
porcine parvovirus
VP2 gene
site directed mutation
eukaryotic expression
