糖基转移酶2基因silD在大肠埃希菌中克隆及表达.docVIP

糖基转移酶2基因silD在大肠埃希菌中克隆及表达 　 作者：洪文荣 曾志红 朱春宝 陈代杰 朱宝泉 【摘要】 从伊纽小单孢菌(Micromonospora inyoensis)扩增参与西索米星生物合成的糖基转移酶基因silD(1181bp)，并分别将其克隆到pUC18和表达载体pET30a上。其开放性阅读框长1173bp，编码含390aa(43.04ku)的多肽链，在大肠埃希菌BL21(DE3)::pETsilD中实现表达。基因silD的碱基序列与gntD(来自M.echinospora)的碱基序列的同源性高达94%。预测的SilD蛋白序列与GntD的同源性为98%。这是继从依纽小单孢菌克隆参与生物合成西索米星的抗性基因srm1(AY661430)、西索米星2脱氧蟹肌醇合成酶基因sisB(DQ250993)和糖基转移酶基因sisZ(DQ250994)后，克隆到的又一新基因。该基因在GenBank的接受号为DQ250992。基因silD的克隆和表达为进一步研究其生物学功能奠定了基础。 【关键词】 糖基转移酶2基因； 伊纽小单孢菌； silD； 克隆与表达 ABSTRACT The gene silD, a novel glycosyl transferase2 gene, was cloned in our laboratory. The DNA fragment containing silD (1185bp) gene (the key gene involved in sisomicin biosynthesis) was isolated from Micromonospora inyoensis and cloned into pUC18 vector. Its ORF is composed of 1,173 bp, encoding 390aa (43.04ku). Sequence analysis verified that silD gene and amino acids sequence showed 94% and 98% identity with gntD (glycosyltransferase gene for gentamicin biosynthesis from M.echinospora), respectively. The prediction of structure of silD suggested it is also a glycosyl transferase protEin. The silD expression vector pETsilD was constructed by fusion of the flanking silD ORF in the pET30a plasmid. The silD protEIn was expressed by IPTG induction in E.coli BL21(DE3):: pETsilD. The successful expression of silD in the E.coli BL21(DE3):: pETsilD will facilitate the further function analysis of the novel gene silD in Micromonospora inyoensis. The silD is a novel gene after cloning the sisomicin resistance gene srm1, glycosyl transferase gene sisZ and 2deoxy scyllo inosose synthase gene sisB from Micromonospora inyoensis. KEY WORDS Glycosyl transferase2 gene; Micromonospora inyoensis; silD; Clone and expression 放线菌中的小单孢菌属微生物能产生重要的氨基糖苷类抗生素，而氨基糖苷类抗生素是临床上重要的抗感染药物。伊纽小单孢菌(Micromonospora inyoensis)产生的西索米星及其相关的衍生物奈替米星被誉为第三代氨基糖苷类抗生素，是氨基糖苷类抗生素中不良反应最少、抗铜绿假单胞菌作用最强、抗钝化酶能力最广的抗生素，世界卫生组织(WHO)将奈替米星列为对人类健康极其重要的抗感染药物。 论文代写 西索米星生物合成途径还未在基因组水平上得到深入研究。采用突变阻断试验，并对突变合成和代谢途径中的中间