应用Bac to Bac 系统表达NDV长春株HN蛋白基因

摘要

Plasmid pKSHNC was cut by BamHI and XbaI. pFast BacI was dealed with as above. HNC was inserted into pFast BacI transposition vector under the control of ocu promoter. Positive recombinant was chosen in LB/AMP+GM culture after transformation in E.coli DH5 α. White bacterial colony was positive recombinant expression vector after transformation in E.coil DH10 Bac and culture in GM, Ka, X-gal and IPTG and selection in blue and white bacterial colony. The positive recombinant expression vector was named of BacmidHNC. Expression of HN protein was examined and identificated by SDS-PAGE and Western-blot after transfection in sf-9 insect cells. The result showed that Bacmid HNC recombinant expression vector were constructed and NDV(Changchun) HN protein gene was expressed in sf-9 insect cells successfully. A special electrophoretic band in SDS-PAGE (74 kDa) was noted and it was proved by Western-blot.%将质粒pKSHNC利用BamHI和XbaI双酶切后回收。将转座载体pFast BacI同样方法酶切、回收。将回收的HNC与pFast BacI连接，使目的基因亚克隆到pFast BacI上的ocu 基因启动子下游的MCS上，然后转化E.coli DH5 α, 以LB/AMP+GM平板筛选阳性重组子，经酶切鉴定，再转化至 E.coli DH10 Bac感受态细胞中，经抗性培养和蓝白斑筛选，挑取白色菌落为阳性重组表达载体，经酶切鉴定后，命名为Bacmid HNC。经小量提取后，转染sf-9细胞，27 ℃培养72 h，应用SDS-PAGE和Western-blot检测和鉴定表达的HN蛋白。结果表明，本研究成功地构建了Bacmid HN重组表达载体，并在sf-9昆虫细胞中表达了NDV长春株HN基因，SDS-PAGE出现一条特异性泳带，约74 kDa，Western-blot结果出现一条杂交带，分子量与之相同，进一步证明构建的重组表达载体表达了NDV长春株HN基因。