Bac-to-Bac杆状病毒表达系统表达小鼠GITRL蛋白并鉴定

目的:利用Bac-to-Bac杆状病毒表达系统表达小鼠糖皮质激素诱导的肿瘤坏死因子受体的配体(G ITRL)蛋白。方法:用EcoRⅠ和SalⅠ双酶切GITRL-PMD18T载体,回收目的基因G ITRL,并定向克隆入穿梭质粒pFastBacHTA中。以pFastBacHTA-G ITRL转化感受态DH10Bac细菌,在DH10Bac细菌内重组pFastBacHTA与杆粒发生转座。筛选阳性克隆,提取重组杆粒,转染Tn昆虫细胞株,获取完整的重组杆状病毒。反复感染Tn细胞,扩增病毒同时表达目的蛋白,用Western blot法进行蛋白鉴定。结果:经核苷酸序列测定及PCR鉴定,成功地获得含GITRL基因的重组杆粒。在杆粒转染的Tn细胞中表现有细胞病变,推断转染成功并获得重组杆状病毒。Western blot法鉴定显示,在Mr20000处有特异性的蛋白条带。结论:利用Bac-to-Bac杆状病毒表达系统成功地表达了小鼠GITRL蛋白,为进一步研究其生物学活性及功能奠定了实验基础。

Expression and identification of mGITRL by Bac-to-Bac baculovirus expression system

AIM:To express the mouse glucocorticoid-induced tumor necrosis factor receptor ligand(GITRL) protein with Bac-to-Bac baculovirus expression system.METHODS: GITRL gene was obtained by double digestion using EcoRⅠ and SalⅠ and cloned into the baculovirus transfer vector pFastBacHTA.Then the pFastBacHTA was transformed into competent 10BacTM E.coli cells.The transposition occurred between pFastBacHTA and bacmid and a recombinant bacmid was obtained.The positive clones were picked out and the recombinant bacmid was isolated,and then complete transfected into Tn cells for producing complete recombinant baculovirus.The baculoviral stock was amplified and the GITRL protein was expressed.RESULTS: The presence of GITRL gene containing the recombinant bacmid was verified by PCR and gene sequencing.The cytopathic effect(CPE) displayed in the transfected Tn cells assumed that the transfection was successful.Western blot analysis showed that the molecular weight of mGITRL protein was about 20 KD.CONCLUSION: The GITRL protein is expressed successfully with Bac-to-Bac baculovirus expression system,which may lay the foundation for further study on biological activity and function of GITRL protein.