Ⅴ型腺病毒纤毛基因的克隆及其在大肠杆菌中的表达

Cloning and expression of adenovirus type Ⅴ fiber in E. coli

目的克隆腺病毒纤毛基因,为构建腺病毒靶向性载体创造条件。方法应用限制性内切酶酶切技术酶切腺病毒骨架质粒pAdEasy1,经多次亚克隆最后完成克隆纤毛基因并构建纤毛基因原核表达载体。用IPTG诱导纤毛蛋白的表达,再用SDS-PAGE和Westernblot对其进行分析。结果成功地克隆纤毛基因。质粒pBS/Fiber经限制性内切酶酶切鉴定及测序证实了其正确性。经质粒pQE/Fiber转化的细菌,在IPTG诱导下可表达一种新的蛋白,并于5h表达量最高。经Westernblot证实,在变性条件下表达蛋白的Mr为62000,在非变性条件下为186000。结论已克隆的纤毛基因能在大肠杆菌中表达,并具有三聚体结构,可用于腺病毒载体靶向性构建。

Aim To clone adenovirus type Ⅴ fiber gene for construction of re-target adenovirus vector. Methods plasmid pAdEasy-1 was digested with restriction endonucleases. The resulting fragment carring fiber gene was then cloned into plasmid Bluescript M13- to generate plasmid pBS/Fiber.To construct fiber protein prokaryotic expression vector pQE/Fiber, the Sph I-Kpn I fragment carring fiber gene from pBS/Fiber was cloned into plasmid pQE.32.The expression of the fiber protein was induced by IPTG,The expressed product was identified by SDS-PAGE and Western blot. Results Both plasmid PBS/Fiber and prokaryotic expression vector pQE/Fiber were successfully constructed. After induction, a new protein band with 62kD appeared on denaturing SDS-PAGE gel,but with 182kD appeared on non-denaturing SDS-PAGE gel and Western blot. Conclusion Expressed fiber is naturely a trimer. The fiber gene can be used for constructing re-target adenovirus vector.