大肠杆菌O85 O-抗原基因簇序列破译和特异分子标识的筛选

Identification of the gene cluster sequence encoding to Escherichia coli O85 antigen and screening of the specific molecular markers

目的测定大测肝菌O85的O-抗原基因簇的序列,对其基因进行功能和进化的分析,筛选并鉴定可用于快速分子分型的特异分子标识。方法利用鸟枪法对大肠杆菌O85的O-抗原基因簇进行测序,生物信息学方法进行序列拼接与分析,确定针对大肠杆菌O85的特异基因并设计引物,PCR方法进行模拟环境样品的检测。结果大肠杆菌O85的O-抗原基因簇序列全长为11 203 bp。发现8个开放阅读框架(orf)并确定功能,分别为:UDP-N-乙酰葡萄糖-2-异构酶基因(orf1),吡喃型UDP-半乳糖变位酶基因(glf),糖基转移酶基因(orf3、orf5、orf6和orf8),O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy)。鉴定出针对大肠杆菌O85的特异基因2个和引物4对。结论本文筛选的特异分子标识和PCR检测方法可用于对环境样品中的大肠杆菌O85进行快速、灵敏的检测。

In the present study, the gene cluster sequences encoding to Escherchia coli O85 antigen are to be identified with the screening of the specific molecular markers for this gene cluster, in which the gene cluster was firsdy amplified by long-range PCR, and then was sequenced by using short-gun sequencing approach. All readings were assembled and analyzed by methods of bioin/ormatics. Primers specific for E. coli O85 antigen were designed and used in PCR to identify the target genes. Furthermore, PCR assay was also used to detect the presence of E. coli O85 antigen in the mimic environmental specimens. It was found that the gene cluster encoding to E. coli O85 antigen contained 8 varieties of genes, including UDP-N- aeetylglueosamine-2-epimerase gene （fl）, UDP-galactopyranose mutase gene （glf）, glycosyl transferase genes （f3, f5, f6, f8）, O-antigen transferase gene （wzx） and O-antigen polymerase gene （wzy）, in which two genes, wzx and wzy and 4 pairs of primers were identified to be specific to E. coli O85 antigen. From the above Observations, it is evident that the specific molecular marker screened and the PCR assay used in the present study may be proved to be valuable for the detection of E. coli O85 antigen from the environmental specimens.