6A8 α—甘露糖苷酶酶解p—nitrophenyl—α—D—mannopyranoside

目的：明确6A8 cDNA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6A8 cDNA,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Western blot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6A8 cDNA碱顺序。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。Western blot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-6A8 cDNA的细胞明显深盂民染空载pCDI及野生型细胞。结论：6A8 cDNA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。

6A8 alpha-mannosidase catalyzes p-nitrophenyl-alpha-D-mannopyranoside

OBJECTIVE: To confirm the alpha-mannosidase nature of the protein encoded by 6A8 cDNA. METHODS: 1) To construct a full-length 6A8 cDNA based on the three cloned DNA fragments by means of gene recombinant technique; 2) To insert the 6A8 cDNA into eukaryotic expression vector pCDI; 3) To transfect the recombinant pCDI-6A8 into COS-7 cells; 4) To characterize the nature of the protein encoded by 6A8 cDNA by means of enzymic activity assay and Western blotting assay. RESULTS: The constructed 6A8 cDNA was the right cDNA in sequence. The enzymetic activity of the homogenate of COS-7 cells transfected with pCDI-6A8 was 3-4 times 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 of 120,000 recognized by mAb 6A8. The band in the cells transfected with pCDI-6A8 cDNA was much darker than that in the cells transfected with the mock or in the wild cells. CONCLUSION: The protein encoded by 6A8 cDNA is a kind of alpha-mannosidase, which belongs to type II alpha-mannosidase.