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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 KSHV-encoded G-protein coupled receptor (vGPCR) gene is an oncogene that plays an important role in KSHV oncogenesis. Previous work revealed that the amino terminus of vGPCR, Y26 and Y28 The thiolation of the two tyrosines is crucial for their tumorigenicity.In this study, the amino-terminal (amino acids 1-49) of vGPCR and its yydd mutant were respectively and organically linked to the Fc fragment of the mouse and named as wt-vG N-mFc and yydd-vG-N-mFc respectively.The HEK293T cells were transfected with the two recombinant plasmids respectively.The recombinant fusion proteins were purified from whole cell lysate and cell culture medium, respectively, and purified by Coomassie blue and blotting Hybridization.Air autoradiography showed that thiolated modification of wt-vG-N-mFc but not yydd-vG-N-mFc.The successful expression and purification of the vGPCR amino-terminal fusion protein containing thiolated tyrosine was The vGPCR tumorigenicity and vG-PCR as the target of clinical therapy laid the foundation for the application.