摘要
为了构建共表达基因Ⅱ型新城疫病毒(NDV)F、HN蛋白的mcDNA载体,试验采用融合PCR技术将合成的Ⅱ型NDV的F基因片段与HN基因片段融合并携带Flag标签,构建pUC-F-HN-Flag基因片段,然后与mcDNA载体MN512A连接构建重组质粒MN512A-pUC-F-HN-opt-Flag,采用菌落PCR扩增与酶切方法对重组质粒进行鉴定;利用阿拉伯糖诱导剂诱导重组质粒产生mcDNA;将重组质粒转染至HEK-293T细胞中,收集细胞总蛋白并通过Western-blot与间接免疫荧光试验检测蛋白表达情况。结果表明:通过F、HN基因片段的扩增与融合成功构建pUC-F-HN-Flag基因片段,菌落PCR扩增与酶切结果证明重组质粒连接成功,经诱导剂诱导重组质粒成功产生mcDNA,Western-blot及间接免疫荧光试验均检测到目的蛋白。说明重组质粒MN512A-pUC-F-HN-opt-Flag构建成功。
The aim of the present study was to construct a micro-circular DNA vector co-expressing F and HN protein of Newcastle disease virus(NDV) type Ⅱ. The F gene fragment of type Ⅱ NDV was fused with the HN gene fragment by fusion PCR technique and the Flag tag was carried to construct the pUC-F-HN-Flag gene fragment. Then the recombinant plasmid MN512 A-pUC-F-HN-opt-Flag was constructed by ligating with mcDNA vector MN512 A. The recombinant plasmid was identified by colony PCR amplification and restriction enzyme digestion and induced to produce mcDNA by arabinose inducer. The recombinant plasmid was transfected into HEK-293 T cells and the total protein was collected. The protein expression was detected by Western-blot and indirect immunofluorescence assay. The results showed that the pUC-F-HN-Flag gene fragment was successfully constructed by amplification and fusion of HN and F gene fragments, and the recombinant plasmid was successfully linked by colony PCR amplification and restriction endonuclease digestion. The recombinant plasmid could successfully produce mcDNA by inducer induction. The expression of the target protein was detected by Western-blot and indirect immunofluorescence. The results indicated that the recombinant plasmid MN512 A-pUC-F-HN-opt-Flag was constructed successfully.
作者
王成宇
姚心茹
刘晶
胡译文
赵维静
姜延龙
王春凤
WANG Chengyu;YAO Xinru;LIU Jing;HU Yiwen;ZHAO Weijing;JIANG Yanlong;WANG Chunfeng(Animal Science and Technology Institute,Jilin Agricultural University,Changchun 130118,China;Jilin Animal Ecological Engineering Research Center,Changehun 130118,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2018年第23期1-5,263,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31602092
31272552
31272541)
国家重点研发计划项目(2017YFD0501000)
“863”国家高技术研究发展计划项目(2013AA102806)
吉林省教育厅“十三五”科学技术研究项目(JJKH20170316KJ)
吉林省科技发展计划项目(20111816)
