摘要
根据NDVNL株的F基因序列，自行设计带有XbaⅠ和XhoⅠ酶切位点的一对引物,应用聚合酶链式反应(PCR)扩增含NDVF基因的PFL质粒,扩增结果与设计相符，大小为630bp。该片段覆盖F基因的2/3的抗原决定簇。对该片段两端酶切后，克隆到融合表达载体pThioHisB中，并转化到大肠杆菌Top10内，利用AMP抗性筛选阳性重组子，并用PCR及双酶切鉴定克隆成功，用IPTG化学诱导表达了F基因。经SDS－PAGE电泳分析，结果在分子量约为35KD处可见到表达的蛋白条带。这与根据核苷酸大小计算的蛋白及融合蛋白之和的大小相符。说明外源片段插入正确表达成功。用HRP标记的兔抗鸡抗体与该条带进行Western－Blot检测，该条带呈阳性反应。进一步证实表达正确。通过蛋白灰度扫描显示表达的融合蛋白占菌体总蛋白的20％。
： We designed a pair of primers by means of the sequence of NDV F gene. Amplification PFL－ DNA was done by PCR and got a 630bp necleotide acid fragment.Then it was directly inserted into the pThiohisB vector DNA. The DNA was digested with XbaI and XhoI and PCR identified. It proved the direction and cloning were correct. It was transferred into expressing host cell E.coli Top10, inducing with IPTG and with temperature sensitiveness. It expressed fusion protein of NDV, which is about 35KD in molecular weight...
出处
《河南畜牧兽医》
2000年第8期6-8,共3页
Henan Journal of Animal Husbandry and Veterinary Medicine
