中文摘要：

脂多糖（LPS）在革兰氏阴性细菌中的功能作用在不同菌株中已有研究,但尚未有对肠杆菌属LPS结构与功能的报道。本研究试图构建能引起肥胖的阴沟肠杆菌B29菌株waaL和waaG基因的缺失突变株,以了解突变体菌株产生的LPS结构与活性与野生菌株产生的LPS的差异。根据同源重组技术原理,利用广宿主自杀性质粒构建成敲除载体pKNG101△waaL和pKNG101△waaG,转化E.coli SM10菌株,并与B29菌株进行双亲杂交,再以抗生素和蔗糖筛选条件筛选waaL和waaG基因缺失突变株;利用多重PCR、ERICPCR、PCR-DGGE、DNA测序结果均证实突变株B29△waaL和B29△waaG构建成功;最后,对野生菌株B29与两个突变株生物学特性的差异进行了比较。银染实验结果表明野生型B29菌株的脂多糖为光滑型结构,突变株的LPS结构明显缺失O抗原,B29△waaG突变株同时还缺失了外部核心部分;用鲎试剂法检测LPS的内毒素活性显示,突变株的内毒素活性与野生型相比有较大幅度下降;B29△waaL突变株的生长速率与野生型相当,而B29△waaG突变株的生长速率则相对降低。本研究成功构建两种内毒素活性下降的突变株,为下一步利用突变株研究B29菌株在肥胖发生中的机制奠定基础。

英文摘要：

The functions of lipopolysaccharide （LPS） in some gram-negative bacteria have been reported in the literatures, but there is no such research on bacteria from the genus of Enterobacter. This study attempts to construct the waaL and waaG deletion mutants of Enterobacter cloacae B29, which was isolated from the feacal sample of a severe obese man. To understand the differences of LPS structure and activity between mutant strain and wild strain. According to principle of homologous recombination, knock-out vectors pKNG101AwaaL and pKNG101AwaaG were constructed by inserting the flanking fragments into the wide host suicide plasmids. Then transformed the knock-out vectors into E. coli SM10 by electroporation and transferred it into B29 by bi-parent mating for recombination. Antibiotics and sucrose were applied for mutant strain selection. Afterwards, the mutant strains were verified by PCR, ERIC-PCR, PCR-DGGE and DNA sequencing methods. The results showed that B29AwaaL and B29AwaaG were successfully constructed. Finally, the differences of biological characteristics such as lipopolysaccharide structure, endotoxin activity, growth rate between the parent strain B29 and two knock-out mutant strains were compared. The result of silver-stained polyacrylamide gels showed that the lipopolysaccharide chemotype of B29 was smooth-type structure, while the size of LPS in mutant strains were obviously smaller than wild-type strain, especially in B29AwaaG. The LAL tests indicated that the endotoxin activity of two mutant strains were lower than the wild type strain. The strain B29AwaaG has a lower growth rate compared with the wild type B29 in the growth curve assay. The constructed waaL and waaG gene deletion mutants which have eliminated endotoxin activity will facilitate further studies on the mechanism of B29 in inducing the obesity.