副猪嗜血杆菌广东分离株外膜蛋白P5基因的克隆及蛋白结构预测

Cloning and of structure prediction of outer membrane protein P5 gene of Haemophilus parasuis

根据GenBank上发表的副猪嗜血杆菌(HPS)外膜蛋白基因的核苷酸序列设计并合成1对特异性引物,从广东省HPS分离株外膜蛋白P5基因中扩增出与预期设计的1116bp大小相符的片段,将扩增产物连接到pMD18-T载体上,进行序列测定和分析。结果表明,克隆出HPS广东分离株的目的基因,核苷酸长为1116bp,共编码371个氨基酸,与已发表的HPS(SH0165)外膜蛋白基因核苷酸序列同源性100%,氨基酸同源性100%。生物学预测分析结果显示,HPS外膜蛋白是一种混合型结构蛋白,含有α-螺旋、β-折叠、β-转角和无规则卷曲,其β-转角和无规则卷曲区域可能形成抗原表位;其N端含有1个信号肽,最佳切割位点在21～22个氨基酸;有15个抗原决定簇;无跨膜区;同源建模分析,未见相似三维结构。

A pair of primers were designed according to Haemophilus parasuis outer membrane protein gene sequences published in the GenBank, and outer membrane protein gene 5 （OMP5） of Haemophilus parasuis isolate was amplified by PCR. The fragment was inserted into the pMD18-T vector,then sequenced and the outer membrane pro- tein secondary structure, antigenic determinants, signal peptide and transmembrane domain of Haemophilus parasuis were predicted with the bioinformatics and molecular biology software. The result showed the OMP5 of HPS GDH15 was 1 116 bp in size,encoding 371 amino acid residues. Compared with the SH0165 outer membrane protein gene published in （;enBank, the homology of nucleotide sequence and amino acid were both 100 ^. The protein structure prediction showed that it was a mixed pattern protein,containing the α-helix,β-pleated sheet,β-turn and random coil. Regions of β-turn and random coil would become the antigenic epitopes. It has 15 antigenic determinants, 1 signal peptide and the best cutting site was between amino acids 21 and 22. But transmembrane domain did not exist in it and no similar three dimensional structure was found. The OMP5 of Haernophilus parasuis GDH15 isolate was conservative,it could be used for the development of genetic engineering vaccine and ELISA method.