应用Bac to Bac系统表达NDV长春株HN蛋白基因

Expression of NDV(Changchun) HN Protein Gene by Bac to Bac System

将质粒 p KSHNC利用 Bam HI和Xba I双酶切后回收。将转座载体 p Fast Bac I同样方法酶切、回收。将回收的 HNC与 p FastBac I连接 ,使目的基因亚克隆到 p Fast Bac I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.coliDH1 0 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 ,2 7℃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 74k Da,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

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.