空肠弯曲菌脂多糖基因ORF6在脂多糖生物合成中作用的研究

Study on The Role of Gene OFR6 from Campylobacter jejuni in The Biosynthesis of Lipopolysaccharide

〔目的〕研究空肠弯曲菌脂多糖基因ORF6在脂多糖生物合成中的作用。〔方法〕通过同源搜索 ,推测基因ORF6的可能功能 ;通过PCR扩增基因ORF6,将基因ORF6插入质粒载体pBluescript中 ,再通过抗卡那霉素弹夹的插入 ,使基因ORF6失活 ;通过自然转移 ,失活的ORF6基因与野生株空肠弯曲菌的常染色体融合而产生突变株 ;通过银染色、蛋白质印迹对突变株脂多糖进行分析 ;结合同源搜索的结果 ,推测基因ORF6的可能功能。〔结果〕ORF6DNA序列与鳗弧菌(Vibrioanguillarum)和脆弱类杆菌 (Bacteroidesfragilis)的转氨酶基因相似 ;通过自然转移得到基因ORF6失活突变株 ;基因ORF6突变株的脂多糖比野生株的脂多糖短 ,基因ORF6的改变影响空肠弯曲菌血清反应。〔结论〕基因ORF6在脂多糖的生物合成中起着重要作用 ;基因ORF6很可能是一种氨基转移酶。

Objective Study the role of Gene OFR6 from C. jejuni in the biosynthesis of lipopolysaccharide.〔Methods〕 By homology searches,a putative function of ORF6 was assigned. A fragment of DNA containing ORF6 was amplified by PCR. To generate C. jejuni knock-out mutant of this gene, the target gene was inserted into a plasmid vector (pBluescript), inactivated by insertion of a Kanamycin resistant cassette and recombined with the chromomsomal DNA by natural transformatnion. The ORF6 mutant LPS structure was analyzed by silver staining and Western blotting.〔Results〕 The ORF6 mutant LPS is 1 kDa smaller than the wild type LPS. The change in the ORF6 mutant prevents the recognition of its LPS by anti-C.jejuni serum.〔Conclusion〕 ORF6 is involved in the biosynthesis of LPS. ORF6 is probably an amino transferase.