中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
8期
702-706
,共5页
张维%孙雯雯%葛新%吕本楠%陈锦英
張維%孫雯雯%葛新%呂本楠%陳錦英
장유%손문문%갈신%려본남%진금영
尿道致病性大肠杆菌%R049基因%外膜蛋白%基因组岛
尿道緻病性大腸桿菌%R049基因%外膜蛋白%基因組島
뇨도치병성대장간균%R049기인%외막단백%기인조도
Uropathogenic Escherichia coli%Gene R049%Outer membrane protein%Genome island
目的 明确从国内分离的尿道致病性大肠杆菌(UPEC) 132中发现的新基因R049的特征及其表达蛋白在菌体内的定位。方法 提取UPEC132染色体DNA，鸟枪法构建文库，高通量焦磷酸法测序，Newbler软件拼接，进行生物信息学分析，确定R049相关片段的特征。提取UPEC132的内膜和外膜蛋白，与其全菌裂解物一起进行SDS-PAGE，用R049重组蛋白抗血清进行Western blot，确定R049表达蛋白的菌体内位置。结果 成功构建UPEC 132染色体文库，获取R049-contig169022 bp，与UPEC536染色体第233074至451502位碱基序列同源性较高，其中含有R049基因的20773 bp片段替代相当于UPEC536染色体上PAIⅢ536的位置，G+C含量为46.97％，两端具有正向重复序列，并与插入元件整合酶基因和thrW tRNA基因相邻，包含25个ORF，命名为R049 -GI。R049基因位于R049-GI的第13个ORF。SDS-PAGE和Western blot分析显示R049基因表达相对分子质量为47.0× 103蛋白是UPEC132的外膜蛋白。结论 R049基因编码细菌的外膜蛋白，是UPEC132通过基因水平转移获得的染色体基因组岛的组成部分。
目的 明確從國內分離的尿道緻病性大腸桿菌(UPEC) 132中髮現的新基因R049的特徵及其錶達蛋白在菌體內的定位。方法 提取UPEC132染色體DNA，鳥鎗法構建文庫，高通量焦燐痠法測序，Newbler軟件拼接，進行生物信息學分析，確定R049相關片段的特徵。提取UPEC132的內膜和外膜蛋白，與其全菌裂解物一起進行SDS-PAGE，用R049重組蛋白抗血清進行Western blot，確定R049錶達蛋白的菌體內位置。結果 成功構建UPEC 132染色體文庫，穫取R049-contig169022 bp，與UPEC536染色體第233074至451502位堿基序列同源性較高，其中含有R049基因的20773 bp片段替代相噹于UPEC536染色體上PAIⅢ536的位置，G+C含量為46.97％，兩耑具有正嚮重複序列，併與插入元件整閤酶基因和thrW tRNA基因相鄰，包含25箇ORF，命名為R049 -GI。R049基因位于R049-GI的第13箇ORF。SDS-PAGE和Western blot分析顯示R049基因錶達相對分子質量為47.0× 103蛋白是UPEC132的外膜蛋白。結論 R049基因編碼細菌的外膜蛋白，是UPEC132通過基因水平轉移穫得的染色體基因組島的組成部分。
목적 명학종국내분리적뇨도치병성대장간균(UPEC) 132중발현적신기인R049적특정급기표체단백재균체내적정위。방법 제취UPEC132염색체DNA，조창법구건문고，고통량초린산법측서，Newbler연건병접，진행생물신식학분석，학정R049상관편단적특정。제취UPEC132적내막화외막단백，여기전균렬해물일기진행SDS-PAGE，용R049중조단백항혈청진행Western blot，학정R049표체단백적균체내위치。결과 성공구건UPEC 132염색체문고，획취R049-contig169022 bp，여UPEC536염색체제233074지451502위감기서렬동원성교고，기중함유R049기인적20773 bp편단체대상당우UPEC536염색체상PAIⅢ536적위치，G+C함량위46.97％，량단구유정향중복서렬，병여삽입원건정합매기인화thrW tRNA기인상린，포함25개ORF，명명위R049 -GI。R049기인위우R049-GI적제13개ORF。SDS-PAGE화Western blot분석현시R049기인표체상대분자질량위47.0× 103단백시UPEC132적외막단백。결론 R049기인편마세균적외막단백，시UPEC132통과기인수평전이획득적염색체기인조도적조성부분。
Objective To investigate the character and location of a novel gene R049 and its expressed protein in uropathogenic Escherichia coli(UPEC) strain 132 isolated in China. Methods The chromosome library of UPEC132 was constructed by a shotgun strategy and the sequence analysis was carried out by a high-throughput pyrophosphate sequencing. Sequence reads were assembled with the Newbler program.The characters of R049-associated specific fragment were analyzed using the bioinformatics methods. Outer and inner membrane proteins of UPEC132 were extracted and then detected by SDS-PAGE and Western blot analysis together with the whole-cell lysates. Results The 169 022 bp contig containing gene R049 was obtained and its sequence was very similar to the chromosome associated sequence of UPEC strain 536. It showed that a 20 773 bp fragment including R049 replaced the pathogenicity island PAI Ⅲ536 of UPEC536 in above 169 022 bp contig. The fragment had a lower GC content (46.97％) and 16 bp direct repeats in two ends. Significantly it also was adjacented to thrW tRNA, insertion element and genes coding integrase. Thus the 20 773 bp fragment was named R049 genome island(R049-GI). There were 25 ORFs in R049-GI, and gene R049 was located in the thirteenth ORF. The results of SDS-PAGE and Western blot revealed gene R049 encoded an outer membrane protein in the size of 47.0× 103. Conclusion Gene R049, encoding an outer membrane protein, was a component part of the genome island in UPEC 132 chromosome acquired by horizontal gene transfer.
