摘要
卡波氏肉瘤相关疱疹病毒(KSHV)编码的G-蛋白偶联受体(vGPCR)基因是一种癌基因,在KSHV致瘤过程中起着重要作用.前期工作揭示vGPCR的氨基端Y26和Y28两位酪氨酸的巯基化作用对其致瘤性至关重要.本研究将vGPCR的氨基端(1～49位氨基酸)以及其yydd突变体分别同鼠Fc片段有机连接并命名为wt-vG-N-mFc和yydd-vG-N-mFc.采用该两种重组质粒分别转染HEK293T细胞,重组融合蛋白分别从全细胞裂解液和细胞培养液中被纯化出来,并被考马斯亮蓝和印迹杂交鉴定.放射自显影表明wt-vG-N-mFc而非yydd-vG-N-mFc具有巯基化修饰作用.含有巯基化酪氨酸的vGPCR氨基端融合蛋白的成功表达和纯化为其将来在研究vGPCR的致瘤性和以vG-PCR为靶定目标的临床治疗中的应用打下了基础.
The Kaposi＇s Sarcoma-associated Herpesvirus （KSHV） encoded G-protein coupled re- ceptor （vGPCR） is an oncogene that is implicated in KSHV associated malignancies. The previous study uncovered that vGPCR incorporates sulfate groups within its N-terminal tyrosine residues （Y26 and Y28） and the tyrosine sulfation is crucial for its tumorigenicity. In this paper, the chimeras fusing the N-terminal （ 1 -49 aa） of vGPCR or its yydd-mutant with mouse Fc fragment were constructed and named wt-vG- N-mFc or yydd-vG-N-mFc accordingly. HEK293T cells were transfected with the constructs and the fu- sion proteins were purified through affinity chromatography from both the whole cell lysates and the media supernatant. The purified wt-vG-N-mFc and yydd-vG-N-mFc were characterized by coommasie staining and western blot individually. The autoradiography test demonstrated that the wt-vG-N-mFc but not the yydd-vG-N-mFc recruits the radioactive labeled sulfate. The successful purification of wt-vG-N-mFc with tyrosine sulfation has established a good foundation for its application in the study of vGPCR tumorigenici- ty and in the clinical therapy for KS targeting vGPCR.
出处
《湖南师范大学自然科学学报》
CAS
北大核心
2011年第6期62-67,共6页
Journal of Natural Science of Hunan Normal University
基金
supported by the National Natural Science Foundation of China(Grant No.81171583 and 3000294)
the Program of Excellent Talent in Hunan Normal University(Grant No.ET31004)
