SEF14菌毛基因操纵子在肠炎沙门氏菌和D-群沙门氏菌的变异分析

【目的】本文旨在探索SEF14菌毛特异性表达于D-群沙门氏菌,特别是肠炎沙门氏菌以及都柏林沙门氏菌的原因。【方法】应用PCR扩增以及序列测定检测了18株鸡白痢沙门氏菌,11株肠炎沙门氏菌以及1株都柏林沙门氏菌标准株中sefA,sefD和sefR基因序列,并分析比对其序列变异。【结果】以11株肠炎沙门氏菌以及1株都柏林沙门氏菌染色体DNA为模板能成功扩增sefA,sefD以及sefR基因;从18株鸡白痢沙门氏菌中均能成功扩增sefA基因,但只有分离于1980年之前的7株分离菌能成功扩增sefD和sefR基因,而另11株1980年后分离菌PCR扩增sefD和sefR基因却无任何产物。比对PCR扩增产物测序结果发现,11株肠炎沙门氏菌以及1株都柏林沙门氏菌株中sefA,sefD以及sefR基因序列和已发表的序列(GenBank登录号为L11008,U07129和AF233854)100%同源;7株鸡白痢沙门氏菌sefD基因测序结果表明,在196位点处发生碱基缺失,造成移码突变,提前于氨基酸残基71位点处产生终止密码子。优化菌毛表达条件,体外抽提和纯化菌毛并进一步试验证明:肠炎沙门氏菌以及都柏林沙门氏菌体外能很好表达SEF14菌毛,但鸡白痢沙门氏菌在相同培养条件下却无任何表达迹象。【结论】SEF14菌毛操纵子亚单位基因sefA,sefD以及调节基因sefR在不同沙门氏菌中的变异情况可能是SEF14菌毛局限性表达的原因之一。

Difference and variation of the sef14 operon gene clusters in S. enteritidis and closed serogroup-D Salmonella

Abstract: Objective] In order to reveal why SEF14 fimbriae are restrictively expressed on strains of serogroup D salmonella, mainly S. enteritidis and S. dublin, the difference and variation of the sef14 operon gene clusters in S. enteritidis and related serogroup-D Salmonella were analyzed. Methods] The genes encoding subunits of sefA, sefD and sefR in S. pullorum, S. enteritidis and S. dublin were amplified by PCR method and then sequenced to analyze the the difference and variation, respectively. Results] The results of PCR amplification showed that prevalence of sefA, sefD and sefR genes in S. enteritidis and S. dublin was 100%. In 18 isolates of S. pullorum, the prevalence of sefA gene was 100%, while the prevalence of sefD and sefR genes was 38.9% (7/18), and 11 strains isolated after 1980s did not contain any gene sefD or sefR. The sequencing data of PCR products revealed that sequences of sefA, sefD and sefR genes in S. enteritidis and S.dublin were identical with those those from NCBI GenBank data which accession number were L11008, U07129 and AF233854, respectively. Interestingly, among the 7 strains of S. pullorum before 1980s, the sefD sequence has a missing base pair at position 196 and caused open reading frame (ORF) shift, resulting in a stop codon (TAG) at position 71 amino acid residual (Leu of TTA at position 214-216 shift into stop codon of TAG at position 215-217). Unlike S. pullorum, all S. enteritidis and S. dublin tested could express SEF14 fimbriae in vitro. Conclusion]Based on the data of the difference and variation of sef14 operon gene clusters between S. enteritidis and S. pullorum, we may explain why SEF14 fimbriae in S. pullorum could not be expressed.