真核表达NDV F基因重组质粒的构建及其在CEF细胞中的转染

Construction of newcastle disease virus(NDV) F gene eukaryotic expression recombination plasmid and its transfection in CEF cells

通过PCR方法自含NDVZF基因的克隆质粒中扩增NDVF基因 ,将其与真核表达载体pcDNA3..1/V5_His_TOPO重组。经核酸内切酶酶切 ,阳性克隆鉴定准确无误 ,核酸序列测定结果与原始基因比较 ,同源性大于 99% ,启始密码和终止密码未出现变异 ,将该重组质粒命名为pcDNANDVZF。将pcDNANDVZF在脂质体作用下转染CEF细胞 ,用间接免疫荧光试验检测 ,结果证明pcDNANDVZF可在CEF细胞中大量表达F蛋白。

The gene NDVF was amplified from cloning plasmid of NDVZ F gene by polymerase chain reaction (PCR),and it was recombinated into eukaryotic expression vector pcDNA3.1/V5-His-TOPO。It was indentified successfully by the restriction enzyme analysis and positive cloning. Nucleotide sequences compared with the relavent sequences of the pattern,the result showed homologous rates were higher than 99 %, there was no differentiation of startup codon and terminate codon, the recombinant plasmid was named pcDNANDVZF.It was transfected into CEF cells with the help of liposome and was detected by indirect immuno- fluorescence experiment, it proved that pcDNA NDVZF was expressed as F protein in CEF cells.