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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。
OBJECTIVE: To express mouse glucocorticoid-inducible tumor necrosis factor receptor ligand (GITRRL) protein using Bac-to-Bac baculovirus expression system. METHODS: The GITRL-PMD18T vector was digested with EcoRI and SalI. The target gene G ITRL was recovered and cloned into the shuttle plasmid pFastBacHTA. Competent DH10Bac bacteria were transformed with pFastBacHTA-G ITRL and the recombinant pFastBacHTA was transposed with the bacmid in DH10Bac bacteria. The positive clones were screened, the recombinant bacmid was extracted and transfected into Tn insect cell lines to obtain a complete recombinant baculovirus. Repeatedly infected Tn cells, amplify the virus while expressing the target protein, using Western blot protein identification. Results: The recombinant bacmid containing GITRL gene was successfully obtained by nucleotide sequence determination and PCR identification. The cytopathic effect was expressed in bacmid-transfected Tn cells, and the transfection was concluded to be successful and a recombinant baculovirus was obtained. Western blot showed that Mr20000 has a specific protein band. Conclusion: The GITRL protein was successfully expressed in Bac-to-Bac baculovirus expression system, which laid the foundation for further study of its biological activity and function.