一种抗topk第74位酪氨酸残基磷酸化的抗体及其制备方法和应用
【技术领域】
[0001] 本发明属于生物和疾病诊断领域，具体涉及一种抗Τ0ΡΚ第74位酪氨酸残基磷酸化 的抗体，本发明还涉及该抗体的制备方法和应用。
【背景技术】
[0002] Τ0ΡΚ，又名，即PDZ连接激酶及淋巴因子激活的杀伤性Τ淋巴细胞源性蛋白激 酶，其作为一种新的丝-苏氨酸蛋白激酶[1，2]，在乳腺癌、结直肠癌、黑色素瘤、肺癌、白血 病等[3]多种肿瘤细胞中呈高表达，并且表达量与乳腺癌和结直肠癌的恶性程度明显相关
[4] J0PK参与了肿瘤细胞周期进程[5-8]、肿瘤细胞转化[9,10]、增殖[11]和凋亡[12,13] 等功能的调控。
[0003] 近年越来越多的研究发现Τ0ΡΚ在肿瘤的临床诊断以及预后预测中发挥了重要作 用。2010年Zlobec I等探讨了在2000例结肠癌病例中Τ0ΡΚ的预后价值，提出Τ0ΡΚ高表达的 病例预后差，同时提出Τ0ΡΚ高表达的病例与酪氨酸激酶抑制剂的耐受有关，Τ0ΡΚ抑制剂的 发现将惠及30-40%结肠癌患者。另外，该研究组还指出Τ0ΡΚ激活相关机制的研究将对克服 酪氨酸激酶抑制剂的耐受有重要意义[14] jeschoolmeester D等认为Τ0ΡΚ是结肠癌的最 有价值的肿瘤标志物之一，与之相关的靶向治疗应逐步展开[15]。也有文献报道Τ0ΡΚ在肺 癌中的表达与Ki67(细胞增殖标记物)和p53密切相关，可能会成为非小细胞肺癌独立的预 后指标[lehShih MC等也认为Τ0ΡΚ通过调控PI3K/AKT/PTEN信号通路影响肺癌细胞的转 移，并可作为肺癌预后的指标和潜在的治疗靶点[17]。另外，Chen F[18]等报道称Τ0ΡΚ表达 水平在人肝癌组织和细胞系中显著上调，在肝癌SMMC-7721细胞中基因沉默Τ0ΡΚ后导致细 胞周期停滞，抑制细胞生长和肿瘤形成，暗示着Τ0ΡΚ可能调节肝细胞的增殖，可能会成为肝 癌有意义的分子靶点和预后标志物。Jae-Hyun等[19]在对81例乳腺癌的全基因组表达谱进 行分析后发现Τ0ΡΚ上调，并且在其细胞的有丝分裂早期呈高表达，可能通过磷酸化Histone H3促进有丝分裂，从而增强肿瘤细胞的增殖。除此之外，Hu F[20]等人发现Myc-E2F1-T0PK 信号通路在恶性淋巴瘤中起着调控作用，Τ0ΡΚ可能会成为淋巴瘤免疫治疗中的靶点。
[0004] 随着研究的深入，Τ0ΡΚ正逐渐成为新的肿瘤发生的标志物和抗肿瘤治疗的靶点。 然而，目前对于Τ0ΡΚ的研究，两个重要的研究瓶颈逐渐显现出来。一是Τ0ΡΚ晶体结构解析困 难，导致其抑制剂领域的研究进行缓慢，阻碍了其用于临床疾病诊断和治疗的发展前景。其 次是Τ0ΡΚ的上游调控机制尚不完全清楚，目前已有报道hDlg[2]，ERK2[9]，c-Myc-E2Fl [20]，P53[21]，E2F-CREB/ATF[22]和 Cdkl/CyclinB[l，23]能与 Τ0ΡΚ 相互作用并调节其功 能。但是，非磷酸化的Τ0ΡΚ表达并不能准确体现Τ0ΡΚ在细胞内的活性，目前仅有研究表明蛋 白激酶Cdkl/Cycl inB和ERK2能在Thr9位点磷酸化激活Τ0ΡΚ，且研究证实Thr9位点磷酸化与 Τ0ΡΚ促进肿瘤发生的关系无关。因此，进一步寻找与肿瘤关系密切的Τ0ΡΚ磷酸化修饰机制， 即Τ0ΡΚ的上游激酶及磷酸化作用位点，并开发相应位点的磷酸化抗体，将有利于扩展Τ0ΡΚ 在临床诊断、治疗方面的实际应用。
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