【摘要】 以大肠杆菌(Escherichia coli)W3350菌株总DNA为模板,根据已报道的几种生物的甘露醇-1-磷酸脱氢酶(mannitol-1-phosphate dehydrogenase)基因(mtlD)序列设计引物,通过PCR扩增到1条长约1.15 kb的特异片断,把该片断连接到pUCm-T载体上进行测序。序列分析结果表明,该基因编码区全长1149 bp,共编码382个氨基酸,与报道的mtlD基因修正序列完全一致。将该基因插入到原核表达载体pET28a(+),IPTG诱导表达后经SDS-PAGE分析,发现在约45 kD处明显比对照多出1条带,凝胶扫描后经Bandscan 5.0软件分析表明,该特异条带的蛋白含量占菌体可溶性总蛋白的72.9%。Western blot检测其为带组氨酸标签的融合蛋白,而质谱分析证实其为甘露醇-1-磷酸脱氢酶。转化子的耐盐能力比对照提高了约20%。该基因提交到GeneBank数据库,返回的接受号为DQ660889。更多还原

【Abstract】 A PCR product of 1.15 kb was obtained from the total DNA of Escherichia coli W3350 strain,by using oligonucleotide primers designed based on the reported sequence of mannitol-1-phosphate dehydrogenase gene of many species.The product was ligated to pUCm-T vector and sequenced.Length of the coding region was 1149 bp and encoded 382 amino acid residues.Its nucleotide acids were the same as the reported sequence.In order to express mannitol-1-phosphate dehydrogenase,the cloned fragment was inserted into the multiple cloning sites of prokaryotic expression vector pET-28a(+),after being induced by IPTG,SDS-PAGE showed an additional band about 45 kD compared to the control.The protein of that band accounted for 72.9% of the total dissoluble protein when detected by Bandscan 5.0.The succedent Western blot and mass spectrometry result indicated that the band was the expressed fusion protein His-mtlD definitely. The salt tolerance level against NaCl of the transformants was 20% higher than that of the control.The gene was accepted by GeneBank with the accession number of DQ660889.更多还原