副猪嗜血杆菌 hhdA克隆、抗原表位预测和表达

摘要

With the reference of the hhdA gene nucleotide sequence, a software was used to design and synthesize a pair of primers and the PCR technology was used to amplify HPS hhdA gene and build its cloning vector.The results showed that nucleotide of the HPS hhdA gene was 1 797 bp in length,compared with the an-nounced hhdA gene nucleotide sequence CP005384.1,the resemblance was 99.8%;the encoded amino acid was 599 aa in length, compared with the announced hhdA gene encoded acid sequece, the resemblance was 99.6%.The bioinformatics analysis of hhdA gene encoded protein was hybrid structural protein, including α-helix regions,β-sheet regions,Beta Angle and random coil.Comprehensive flexible Regions,hydrophobicity Plot, antigenic index and surface probability plot predicted the possible advantageous B cell epitope antigen epitope of hhdA protein.The hhdA gene was cloned into pET-32a(+) vector to generate the recombinant expressing plasmid pET-32a(+) -hhdA,then pET-32a (+)-hhdA was transformed into the host cell BL21 with the induction of IPTG,the result of SDS-PAGE showed that expression of fusion protein was 80 kD.All this Lays the foundation for further research of the biological function of HPS hhDA,antibody preparation and development of genetic engineering products of hhdA gene.%根据GenBank中的副猪嗜血杆菌hhdA的基因序列，设计并合成l对特异性引物，对从江西分离的HPS的hhdA基因进行扩增、克隆、测序、生物信息学分析和原核表达。测序结果表明，扩增的目的基因大小为1797 bp，共编码氨基酸599个氨基酸。与GenBank上公布的ZJ0906株（ CP005384．1）中对应的核苷酸序列同源性为99．8％，氨基酸同源性99．6％。生物信息学分析表明，HPS hhdA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；综合柔性区域、亲水性位点、抗原指数和表面可能位点预测了hhdA蛋白可能的B细胞抗原表位的优势表位。将目的基因转入原核表达载体pET－32a（＋）构建重组原核表达质粒pET－32a（＋）－hhdA，将重组质粒转化到BL21（ DE3）中，进行IPTG诱导表达，经过SDS－PAGE电泳结果显示表达的融合蛋白约80 kD。为进一步研究HPS hhdA生物学功能及抗体制备和开发hhdA基因工程产品奠定了基础。