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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pAdEasy1,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 Cloning of adenovirus cilia gene, to create conditions for the construction of adenovirus targeting vector. Methods Restriction endonuclease digestion technique adenovirus backbone plasmid pAdEasy 1, after multiple subcloning cloning cilia gene complete and construct ciliated gene prokaryotic expression vector. The expression of the protein was induced by IPTG and analyzed by SDS-PAGE and Western blotting. Results The cloning gene was successfully cloned. Plasmid pBS / Fiber restriction enzyme digestion and sequencing confirmed its correctness. The bacteria transformed with plasmid pQE / Fiber expressed a new protein induced by IPTG and expressed the highest level at 5h. Western blot confirmed that Mr expressed 62,000 under denaturing conditions and 186,000 under non-denaturing conditions. Conclusion The cloned cili gene can be expressed in E. coli and has a trimeric structure, which can be used for targeted construction of adenovirus vector.