Cloning, expression and identification of a gene CgFUT5 associated with Lewis antigen synthesis of Oyster norovirus receptors
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摘要: Lewis抗原被认为是诺如病毒特异性结合受体，作为诺如病毒传播载体，牡蛎中也存在着类Lewis抗原，但牡蛎合成这种碳水化合物的途径尚未阐明。为解析牡蛎中诺如病毒受体类Lewis抗原的合成路径，利用cDNA末端快速扩增 (Rapid amplification of cDNA ends, RACE) 技术克隆得到太平洋牡蛎 (Crassostrea gigas) 的CgFUT5基因全序列并进行生物信息学分析，通过实时荧光定量聚合酶链式反应 (RT-qPCR) 分析其在5种组织中的表达情况。构建原核表达质粒转化大肠杆菌 (Escherichia coli) 实现异源表达，并通过免疫印迹法 (Western blot) 鉴定免疫原性。克隆得到了具有1 173 bp开放阅读区的CgFUT5基因cDNA序列，系统发育树显示CgFUT5基因与多个物种具有合成Lewis抗原功能的岩藻糖基转移酶基因遗传学关系较近。重组CgFUT5蛋白可在大肠杆菌中过量表达，且表达的重组CgFUT5蛋白与抗人FUT5抗体及抗6×His标签抗体均能特异性结合。研究发现CgFUT5基因在牡蛎鳃组织中大量表达，CgFUT5蛋白与人FUT5蛋白具有相似的免疫原性，推测牡蛎中存在着类Lewis抗原的合成通路，并且调控牡蛎类Lewis抗原合成的基因还具有组织表达差异性。Abstract: Lewis antigen is regarded as a specific binding receptor for norovirus, and Lewis-like antigen is also present in oysters as a vehicle for norovirus transmission, but the pathway for synthesis of this carbohydrate in oysters has not been elucidated. To clarify the pathway of norovirus receptor-like Lewis antigen synthesis in oysters, we cloned the CgFUT5 gene from the Pacific oyster (Crassostrea gigas) genome and analyzed it for expression in five tissues. The full sequence of CgFUT5 gene was obtained by rapid amplification of cDNA ends (RACE) and bioinformatically analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). A prokaryotic expression plasmid was constructed to transform Escherichia coli for heterologous expression, and immunogenicity was identified by immunoblotting (Western blot). The cDNA sequence of CgFUT5 gene with 1 173 bp open reading region was obtained by cloning, and phylogenetic tree shows that CgFUT5 gene was genetically related to the rockweed glycosyltransferase gene that has the function of synthesizing Lewis antigen in several species. The recombinant CgFUT5 protein could be overexpressed in E. coli, and the expressed recombinant CgFUT5 protein specifically bound to both anti-human FUT5 antibody and anti-6×His tag antibody. To sum up, CgFUT5 gene was successfully cloned and found to be abundantly expressed in oyster gill tissue, and CgFUT5 protein had similar immunogenicity to human FUT5 protein. It was hypothesized that a Lewis-like antigen synthesis pathway exists in oysters, and the genes regulating Lewis-like antigen synthesis in oysters also have differential tissue expression.
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Key words:
- Crassostrea gigas /
- CgFUT5 gene /
- Clone /
- Tissue expression /
- Prokaryocyte expression
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图 1 太平洋牡蛎FUT5基因全长及氨基酸序列
Figure 1. Full length and amino acid sequence of FUT5 gene in C. gigas
图 4 CgFUT5 基因在太平洋牡蛎 5 种组织中的相对表达量
注：不同字母代表组间极显著性差异 (P<0.01)。
Figure 4. Relative expression of CgFUT5 gene in five tissues of C. gigas
Note: Different letters represent extremely significant differences among the groups (P<0.01).
图 5 CgFUT5重组蛋白的SDS-PAGE电泳分析
Figure 5. Analysis of CgFUT5 recombinant protein by SDS-PAGE electrophoresis
图 6 以鼠抗 6×His 标签单克隆抗体为一抗分析CgFUT5 表达蛋白
Figure 6. Expression of CgFUT5 protein analyzed by mouse anti-6×His labelled monoclonal antibody as primary antibody
图 7 以兔抗人 FUT5 单克隆抗体为一抗分析CgFUT5 表达蛋白
Figure 7. Expression protein of CgFUT5 analyzed by rabbit anti-human FUT5 monoclonal antibody as primary antibody
表 1 实验中所用引物
Table 1. Primers used in this experiment
引物
Primer序列 (5'—3')
Sequence (5'−3')用途
FunctionFT5-1 CCAGAGCCAAAAACCTCACTC 中间片段克隆 RT5-1 TCCCAGCGAAATCTACTTCC RRT5 GATTACGCCAAGCTTGTAATGACTGGACACGACACTGTTCTTG RACE FRT5 GATTACGCCAAGCTTACGCATCTCCTGAAGAATTGGCTAAGG Q-FT5-A TCTGTATTCTGTAAGGCCGGAGTGG 荧光定量组织表达分析 Q-RT5-A AGTTTCGGGACAATGGGATTTCTCG F-actin CTGTGCTACGTTGCCCTGGACTT R-actin TGGGCACCTGAATCGCTCGTT 
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