6A8α-甘露糖苷酶酶解p-nitrophenyl-α-D-mannopyranoside

6A8α-mannosidase Catalyzes p-nitrophenyl-α-D-mannopyranoside

目的明确6A8cDNA编码蛋白质的α-甘露糖苷酶性质。方法利用基因重组技术将克隆到的3个DNA片段拼接成完整6A8cDNA将其插入到真核表达载体pCDI转染COS-7细胞,用酶活性检测与Westernblot对瞬时表达的蛋白质的性质进行鉴定。结果拼接后的cDNA片段经测序完全符合6A8cDNA碱基顺序。转染重组表达载体pCDI-6A8的COS-7细胞的匀浆对p-nitrophenyl-α-D-mannopyranoside的酶解作用为转染空载体pCDI的细胞及野生型细胞的3～4倍,且这种酶解作用不能被swainsonine所抑制。Westernblot检测显示在相对分子质量(Mr)120000处有一明显蛋白带,此带的显影于转染pCDI-6A8cDNA的细胞明显深于转染空载pCDI及野生型细胞。结论6A8cDNA编码的蛋白质确为一种α-甘露糖苷酶,属于Ⅱ类α-甘露糖苷酶。

Objective To confirm theα-mannosidase nature of the protein encoded by6A8cDNA.Meth-ods 1)To construct a full-length6A8cDNA based on the three cloned DNA fragments by means of gene recombinant technique;2)To insert the6A8cDNA into eukaryotic expression vector pCDI;3)To transfect the recombinant pCDI -6A8into COS-7cells;4)To characterize the nature of the protein encoded by6A8cDNA by means of enzymetic activity assay and Western blotting assay.Results The constructed6A8cDNA was the right cDNA in sequence.The enzymetic activity of the homogenate of COS-7cells transfected with pCDI -6A8was3-4times higher than that of the cells transfected with the mock or the wild cells.The enzymetic reaction could not be inhibited by swainsonine.Western blot showed a band of120000recognized by mAb6A8.The band in the cells transfected with pCDI -6A8cDNA was much darker than that in the cells transfected with the mock or in the wild cells.Conclusion The protein encoded by6A8cDNA is a kind ofα-mannosidase,which belongs to typeⅡα-mannosidase.