猪源Bb SC-SN株的分离鉴定及其皮肤坏死毒素(DNT)全基因序列分析

Identification of an Bb SC-SN isolated from Sichuan and sequence analysis of DNT gene

【目的】了解当前支气管败血波氏杆菌（Bb）地方菌株皮肤坏死毒素（DNT）基因的变异特点。【方法】利用分离培养、PCR及16SrRNA扩增片段同源性分析,对采自四川遂宁某规模化猪场有明显萎缩性鼻炎症状的鼻拭子进行病原分离鉴定,并对分离株的DNT基因进行克隆和序列分析,对其抗原域、氨基酸二级结构、特定区域位于蛋白质表面可能性等参数进行预测,分析其B细胞抗原表位。【结果】成功分离得到一株支气管败血波氏杆菌,命名为支气管败血波氏杆菌SC-SN株。分离株DNT基因序列全长4 357bp,与其他11株菌DNT基因的核苷酸序列同源性为93.1%~100.0%,但由于其356位插入碱基A,使得该序列仅编码1 426个氨基酸,与上述11株DNT基因编码的氨基酸序列同源性仅为12.8%~47.0%。遗传进化树显示,SC-SN分离株与S798日本株和未知1株的亲缘关系最近。软件预测结果显示,Bb SC-SN株DNT基因编码氨基酸B细胞抗原表位发生较大变化。【结论】经鉴定,分离株确为支气管败血波氏杆菌,且SC-SN株的DNT基因序列变异较大,第356位插入的碱基A,对其氨基酸序列、ORF及B细胞抗原表位分布产生了较大的影响。

【Objective】This study investigated the variation characteristics of Bordetella bronchiseptica DNTgene in Sichuan.【Method】A Bordetella bronchiseptica strain was identified from swine nose swab with respiratory disease from a pig farm in Suining,Sichuan by cultivation,PCR and 16 SrRNA homology analysis.Then,DNT of the strain was cloned,the sequence,antigenicity,amino acid secondary structure,certain location surface accessibility in protein and B cell epitopes were analyzed.【Result】A Bordetella bronchiseptica strain was separated successfully and named Bb SC-SN.The sequence analysis showed that the length of DNT was 4 357 bp,and it coded 1 426 amino acids with A inserted in the 356 th position.The DNTsequence of SC-SN strain shared 93.1%-100.0% homology with other 11 strains,but the homologyof amino acid sequences was 12.8%-47.0%.Phylogenetic tree showed that DNTof SC-SN strain had closest relationship to S798.The B cell epitopes of Bb SC-SN DNTamino acid changed greatly according to model prediction.【Conclusion】The isolated SC-SN strain was Bordetella bronchiseptica,and its DNTvaried greatly.The base A at the 356 th position had great influence on amino acids,ORFs and B cell epitopes.of amino acid sequences was 12.8%-47.0%.Phylogenetic tree showed that DNTof SC-SN strain had closest relationship to S798.The B cell epitopes of Bb SC-SN DNTamino acid changed greatly according to model prediction.【Conclusion】The isolated SC-SN strain was Bordetella bronchiseptica,and its DNTvaried greatly.The base A at the 356 th position had great influence on amino acids,ORFs and B cell epitopes.