太平洋牡蛎中类FUT10蛋白的原核表达及免疫印迹鉴定

Prokaryotic expression and western blot identification of FUT10 protein in Crassostrea gigas

中文关键词:  太平洋牡蛎  类FUT10基因  原核表达

英文关键词:Crassostrea gigas  FUT10-like gene  prokaryocyte expression

中文摘要:

      目的 获取太平洋牡蛎类α1,3岩藻糖基转移酶10 (α1,3 fucosyl-transferase 10-like, FUT10-like)蛋白并探究与人FUT10的免疫相似性。方法 本研究基于课题组前期克隆完整的类FUT10基因cDNA, 依照大肠杆菌表达蛋白的密码子偏爱性, 利用GenSmart Optimization在线优化软件对类FUT10基因开放阅读框序列进行优化并合成, 亚克隆至载体pET-28a (+), 得到重构质粒pET-OFu3, 转入大肠杆菌BL21 (DE3), 通过异丙基-D-硫代半乳糖苷(isopropyl-D-thiogalactoside, IPTG)诱导后获取目的蛋白, 进行蛋白质印迹法(Western blot)鉴定。结果 在37 ℃、IPTG终浓度为1.0 mmol/L诱导4 h后, 出现蛋白分子量大小约为55 kDa的条带。该蛋白条带与抗6×His(组氨酸标签)抗体、抗人FUT10抗体均能发生特异性结合。结论 优化后的类FUT10蛋白与人体转移酶FUT10具有相似的免疫原性, 类FUT10蛋白的可溶性表达为探究太平洋牡蛎体内Ⅱ型血型抗原合成路径提供研究基础。

英文摘要:

      Objective To obtain Crassostrea gigas α1,3 fucosyl-transferase 10-like (FUT10-like) protein and explore the immune similarity with human FUT10. Methods In this study, based on the previously cloned FUT10-like gene cDNA, according to the codon preference of the expressed protein in Escherichia coli, the open reading frame sequence of FUT10-like gene was optimized and synthesized by GenSmart Optimization software, and subcloned into vector pET-28a (+) to obtain the reconstructed plasmid pET-OFu3 and transferred into Escherichia coli BL21 (DE3). The protein was obtained after induction by isopropyl-D-thiogalactoside (IPTG). FUT10-like proteins were identified by Western blotting. Results After induction for 4 h at 37 ℃ and the final concentration of IPTG was 1.0 mmol/L, a band about the size of 55 kDa appeared. The target protein band could specifically bind to anti-6×His tag antibodyand anti-human FUT10 antibody. Conclusion The optimized FUT10-like protein has similar immune resistance to human FUT10. The soluble expression of FUT10-like protein provides a research basis for exploring the synthetic pathway of type Ⅱ histo-blood group antigens in Crassostrea gigas.