兽疫链球菌透明质酸合成相关基因hasB的克隆以及性质研究

Molecular Cloning and Characterization of hasB from an Operon Required for Hyaluronic Acid Synthesis in Streptococcus equi subsp. zooepidemicus

透明质酸是链球菌荚膜的主要组成部分,有着重要的生理功能。UDP-葡萄糖脱氢酶(HasB)是透明质酸合成中的一个关键酶,而C类链球菌的UDP-葡萄糖脱氢酶编码基因(hasB)尚未被克隆。通过hasB基因的上下游序列设计引物从兽疫链球茵的基因组中克隆出一段序列,测序结果显示其包含一个由1206个碱基组成的开放阅读框,所编码的蛋白序列同化脓链球菌和乳链球菌的UDP-葡萄糖脱氢酶蛋白序列分别有63.1%和70.6%的相似性。将这段基因置于T7启动子下,并在大肠杆菌中进行表达,能够得到一个约47kDa的蛋白,酶活测定显示其具有UDP-葡萄糖脱氢酶活性。这些结果表明所克隆的基因是兽疫链球菌的UDP-葡萄糖脱氢酶编码基因。

UDP-glucose dehydrogenase (HasB) is an important enzyme in the formation of hyaluronic acid, which forms the capsule of Streptococci. The hasB of group C Streptococci has not been cloned and characterized. The putative UDP-glucose dehydrogenase gene (hasB) was cloned and sequenced. An open reading frame of 1206 base pairs was identified. The hasB gene product was shown to have 63 .1 % similarity with HasB of Streptococcus pyogenes and 70.6 % with that of Streptococcus uberis . In order to show that hasB expression correlated with UDP-glucose dehydrogenase activity, the hasB gene was cloned under control of the T7 promoter. Hyperexpression of hasB resulted in a protein of approximately 47kDa, high levels of UDP-glucose dehydrogenase activity were observed. These data demonstrated that hasB encodes the UDP-glucose dehydrogenase of group C Streptococci.