红斑丹毒丝菌SpaA抗原基因的克隆、序列分析及蛋白结构预测

Cloning and sequence analysis of SpaA gene of Erysipelothrix rhusiopathiae and prediction of protein structure

参照红斑丹毒丝菌表面抗原A(SpaA)基因的核苷酸序列合成1对引物,对我国生产用红斑丹毒丝菌CVCC43005的SpaA全基因进行了PCR扩增,扩增产物连接到pMD18-T载体上,进行序列测定和分析。结果表明,SpaA基因全长1 881bp,含有1个开放性阅读框,编码626个氨基酸。与已提交的红斑丹毒丝菌血清型1a、1b、2a、2b、5、8、9、12、15、16、17、N型的氨基酸同源性为97.5%～100%,血清型4、6、11、19、21的氨基酸同源性为52.8%～55.2%。与已提交的丹毒丝菌血清型18的氨基酸同源性为57.5%～58.0%。采用Chou-Fasman和Karplus-Schulz方案预测蛋白质的二级结构和柔性区域;运用Kyte-Doolittle方案预测氨基酸的亲水性,按Jame-son-Wolf方案预测抗原指数,利用Emini方案预测蛋白质的表面可及性。对预测结果综合分析,推测最有可能的B细胞表位位于N端的59～64、85～91、174～186、193～201、212～215、221～231、266～275、278～288、291～308、314～327、329～349、356～376、403～456、508～516、528～537、568～576和607～626。

According to the sequence of SpaA gene of Erysipelothrix rhusiopathiae,a pair of primers was synthesized to amplify the full-length SpaA gene of strain CVCC43005. The PCR product was cloned into pMD18-T vector for sequencing. Sequence analysis showed that the complete ORF of SpaA gene was 1881 bp,encoding 626 amino acid. Compared with SpaA of serovar la,lb,2a,2h,5,8,9,12,15,16,17 and N,SpaB of the serovar 4,6,11,19 and 21, SpaC of the serovar 18,the deduced amino acid homology was 97.5% to 100% ,52.8% to 55, 2% ,57.5% to 58%, respectively. The secondary structure and the flexibility plot of SpaA were predicted by the algorithm of Chou-Fas-man and Karplus-Schulz based on SpaA sequence, and the hydrophilicity plot, the antigenic index and the surface probability plot were predicted by the methods of Kyte-Doolittle,Jameson-Wolf and Emini, respectively. The B cell epitopes of SpaA by these predicted results was analyzed,which are most likely localized in oradjacent to its N-temi- nal No. 59 64,85-91,174-186,193-201,212-215,221-231,266-275,278-288,291 308,314-327,329-349,356-376,403- 456,508-516,528-537,568-576 and 607-626.