太平洋牡蛎(Crassostrea gigas)类α-1,2-岩藻糖基转移酶的密码子优化与原核表达

Codon Optimization and Prokaryotic Expression of Fucosyltransferase 2 Like Protein from the Oyster (Crassostrea gigas)

中文关键词: 太平洋牡蛎  类FUT2基因  密码子优化  原核表达

英文关键词: Crassostrea gigas  FUT2 like gene  Codon optimization  Prokaryocyte expression

中文摘要:

      牡蛎消化组织内存在的类A型血型组织抗原是其特异性富集诺如病毒的主要原因，FUT2(Fucosyltransferase 2，α-1,2-岩藻糖基转移酶)是A型血型组织抗原合成的关键酶。本研究在前期克隆了太平洋牡蛎(Crassostrea gigas)类FUT2基因cDNA全长的基础上，根据大肠杆菌密码子偏爱性优化并合成了类FUT2基因，插入原核表达载体pRSET A构建pRSET-mof，将其转化大肠杆菌BL21，用异丙基-β-D-硫代半乳糖苷(IPTG)诱导。经SDS-PAGE分析显示，在37℃、IPTG终浓度为0.8 mmol/L的条件下，诱导4 h后出现大小约为46 kDa的特异性目的条带。利用His亲和层析柱纯化及超滤管浓缩目的蛋白，得到单一条带，说明纯化效果良好。Western blot分析显示，目的蛋白与抗6×His标签单克隆抗体、抗人FUT2单克隆抗体均能发生特异性反应，表明优化后的太平洋牡蛎类FUT2基因在大肠杆菌系统中成功表达。本研究结果为今后研究太平洋牡蛎类FUT2基因的功能，进一步探索牡蛎特异性富集诺如病毒的分子机理奠定了基础。

英文摘要:

      Norovirus is the most common cause of acute gastroenteritis and is responsible for substantial morbidity and mortality worldwide. The oyster is considered to be one of the major vehicles for foodborne disease caused by Norovirus. The A-like histo-blood group antigens (HBGA) in oyster digestive tissues is a major reason for Norovirus specific bioaccumulation in oyster, and α-1,2- fucosyltransferase (FUT2) is one of the key enzymes required in the mammal HBGA synthesis. Based on our previously cloned the full-length cDNA of FUT2-like gene in oysters (Crassostrea gigas) (GenBank ID: KJ184342), we re-designed and artificially synthesized the open reading frame of FUT2-like gene by adopting the codons preferentially used in Escherichia coli without any change of the amino acid sequences. The synthesized FUT2-like mutant gene was inserted into the pRSET A to construct prokaryotic expression plasmid pRSET-mof. The recombinant plasmid was identified by enzyme digestion and sequencing, and then expressed in E. coli BL21 (DE3) cells through isopropyl-β-thiogalactop­yranoside (IPTG) induction. SDS-PAGE analysis showed that inducing the BL21 (DE3) cells at 37℃ in 0.8 mmol/L of IPTG for 4 hours were the optimal conditions for expression of the recombinant fusion protein. The molecular mass of the expressed protein product was about 46 kDa, which was consistent with the prediction. The recombinant protein was purified and concentrated by Ni-chelating affinity chromatography and the ultrafiltration. SDS-PAGE analysis showed a single band (about 46 kDa), which means a good purification result. Western blot analysis showed that the recombinant fusion protein could be specifically combined with mouse anti-His-Tag Mab and mouse anti-human FUT2 Mab. So the expressed protein was confirmed to be the aimed protein. These findings would provide a basis for further studies on the function of FUT2-Like gene of oyster and the probable molecular mechanism of accumulation of NoV by oysters.