摘要
目的构建携带N末端Strep(NS)蛋白标签的表达载体;在表达载体中构建衣原体RNA聚合酶亚基重组蛋白并表达。方法采用PCR的方法,通过引物引入NS蛋白标签和新的多克隆位点替代p ET21c-DH质粒中原有的T7蛋白标签和多克隆位点,选择新引入的Not I酶切位点进行PCR产物的环化自连,转化DH-5α细菌后筛选阳性菌株,PCR法和基因测序法鉴定新构建的表达载体;在新构建表达载体的Bam H I和Sal I位点之间分别插入衣原体RNA聚合酶核心酶的α、β、β'亚基,获得表达NS-α、NS-β、NS-β'融合蛋白的表达载体,转化表达菌株Arctic Express,筛选阳性表达菌株,并用考马斯亮蓝染色、Western blot等法鉴定融合蛋白的表达情况。结果成功构建了携带NS蛋白标签的p ET21c-NSMCS载体,并成功将其应用于衣原体RNA聚合酶核心酶亚基融合蛋白的构建及表达。结论获得稳定表达NS-α、NS-β、NS-β'融合蛋白的表达载体,为研究衣原体基因转录调控奠定了良好的基础。
Aims To construct the N-terminal Streptagged( NS-tagged) fusion protein expression vector,and to apply the vector to express NS-tagged fusion proteins of Chlamydia RNA polymerase subunit. Methods By using PCR method,NS fusion protein tag and a new multiple cloning sites( MCS) were inserted into p ET21c-DH plasmid by primers to replace the original T7 protein tag and MCS. The newly introduced Not I cutting site was chosen for self-ligation of PCR product. Then,the cyclized PCR product was transformed into DH-5α competent cells. The positive clones were selected by PCR and sequencing. To get NS-tagged fusion proteins of chlamydial RNA polymerase subunits,the α,β and β' subunits were inserted between Bam H I and Sal I cutting sites of the newly constructed expression vector. Then,the NS-α,NS-β and NS-β' expression vectors were transformed into Arctic Express expression cells. The fusion protein expression statuses of transformed cells were identified by Commassie blue staining and Western blot. Results The NS-tagged fusion protein expression vector p ET21c-NS-MCS was successfully constructed,and NS-α,NS-β and NS-β'fusion proteins were obtained by using this newly constructed expression vector. Conclusions In this project,we constructed an NS-tagged fusion protein expression vector and applied it to express NS-α,NS-βand NS-β' fusion proteins. Our study can lay a solid foundation for the study of transcriptional regulation of Chlamydia genes.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2016年第1期98-102,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 31370209
31400165)
南通大学研究生科研创新计划项目(No 13025166)
"青蓝工程"资助项目
