中文摘要：

目的：酪氨酸蛋白激酶NOK／STYKl具有很强的促肿瘤形成和转移能力，被认为是很有前途的肿瘤治疗靶点。由于NOK含有一个跨膜区，且富含疏水性氨基酸，其表达和纯化非常困难，直接影响了对其功能及相关分子机理的深入研究。本研究目的是获得可溶的且纯度较高的NOK胞内区融合蛋白ANOK（AA：49—422），为后续抗体的制备和功能研究奠定重要基础。方法：含有△NOK基因的原核表达载体，转入E-coliBL21中，IPTG诱导蛋白表达，通过亲和层析获得可溶的△NOK融合蛋白。融合蛋白经凝血酶酶切后，凝胶过滤层析分离标签蛋白获得z~NOK蛋白。同时，我们还通过Bac-to-Bac系统获得含有ANOK基因的杆状病毒，感染sO细胞，尝试在真核细胞中表达目的蛋白。结果：通过在sf9昆虫细胞和大肠杆菌表达系统中盐浓度等各种条件的摸索，首次获得了可溶的且纯度较高的NOK胞内区融合蛋白（ANOK—GST）和一定量去除标签的ANOK蛋白。本研究中与大肠杆菌相比，昆虫细胞并不适合△NOK的纯化。结论：我们建立了一套优化的NOK蛋白表达和纯化体系，从而为后续抗体制备和各种体内外生化实验等功能研究奠定基础，为研究NOK在肿瘤中的作用和药物筛选创造条件。同时丰富了整个RTKs家族作用机制的探索，进一步促进了以RTKs为靶点的治疗手段在临床上的应用。

英文摘要：

Objective： NOK/STYK1, a novel oncogene with kinase-domain, plays important roles in many kinds of tumors and is considered to be a promising target for cancer therapy. Since it contains a single transmembrane domain and is rich of hydrophobic amino acids, its soluble expression and purification are extremely difficult. Here we tried multiple expression systems to lay an important foundation for its further study, such as preparation of specific antibody and structure analysis. Methods： The recombinant NOK expression plasmid was transformed into the E.coli BL21. After induction of IPTG, soluble recombinant protein was lysed and then purified through affinity chromatography. GST tag was cut with thrombin then separated by gel-filtration chromatography. Besides, we attempted to get soluble NOK protein in sf9 cells by infection with recombinant baculovirus which was got through Bac-to-Bac system. Results： The soluble recombinant protein ANOK-GST （AA： 49-422） as well as ANOK without tag was firstly successfully purified by optimization of affecting factors in insect cell and E.coli. Also, it seems that sf9 cells were not suitable for NOK purification compared with E.coli in our research. Conclusion： We established a set of procedures to get soluble recombinant intracellular domain of NOK protein （ANOK-GST） for the preparation of antibodies and in vitro biochemical function studies. This method can be useful for the study on the role of NOK in tumor development and drug screening. Furthermore, this study can extend our understanding on the function mechanisms of RTKs family and promote their clinical applications.