【作者】 丁向明；

【导师】 吴士良； 周迎会；

【作者基本信息】 苏州大学 ， 生物化学与分子生物学， 2004， 硕士

【摘要】 目的：原核表达多肽：N-乙酰氨基半乳糖转移酶2全长编码序列；模拟多肽：N-乙酰氨基半乳糖转移酶2蓖麻蛋白样结构域三维结构。方法：用PCR技术从pDONR201-T2得到ppGalNAc-T2全长编码序列，亚克隆至原核表达载体pGEX-4T-1，转化BL21大肠杆菌，IPTG诱导表达该融合蛋白；利用SWISS-MODEL服务器对多肽：N-乙酰氨基半乳糖转移酶2蓖麻蛋白样结构域同源建模并通过结构比对寻找其活性位点。结果：构建了原核表达重组载体pGEX-4T-1-T2，SDS-PAGE检测到一分子量为90.7KD融合蛋白的表达，与理论计算值相符；ppGalNAc-T2蓖麻蛋白样结构域二级结构主要为β链，采用的是β-三叶草折叠方式，ASN4、ASP7、ASN28三个氨基酸可能为其糖结合活性位点。结论：本研究成功表达了ppGalNAc-T2全长编码序列，为进一步测定其酶活性打下基础；得到了ppGalNAc-T2蓖麻蛋白样结构域的模拟三维结构，为该结构域在酶活性中可能的作用提供了依据。更多还原

【Abstract】 Objective: To express the full-length encoding sequence of human polypeptide N-Acetylgalactosaminyltransferase 2 in Escherichia coli BL21; to simulate the 3D structure of ricin-like domain of ppGalNAc-T2. Methods: The cDNA gene encoding ppGalNAc-T2 was subcloned from plasmid pDONR201-T2 into prokaryotic expression vector pGEX-4T-l, resulting in the recombinant plasmid pGEX-4T-l-T2, which was subsequently transformed into Escherichia coli BL21. IPTG was used to induce the expression of the target gene; Exploit SWISS-MODEL, an automated protein homology-modeling server, to simulate the 3D structure of the ricin-like domain of ppGalNAc-T2 and predict the possible active site through comparing with the temple protein. Results: The plasmid pGEX-4T-l-T2 was reconstructed successfully and a new added Mr 90.7xl03 fusion protein was detected by 12% SDS-PAGE. The fold of the predicted structure is β -trefoil and the second structure is mainly β strand and ASN4, ASP7, ASN28 may constitute the sugar-binding site, which may play an essential role in the enzyme activity of ppGalNAc-T2. Conclusion: We have successfully expressed the ppGalNAc-T2 gene in Escherichia coli BL21, which establishes a foundation for the further research in detecting the enzyme activity of ppGalNAc-T2; We have obtained thepredicted 3D structure of the ricin-like domain of ppGalNAc-T2, which provides evidence for the role played by such domain in the enzyme activity of ppGalNAc-T2.更多还原