副鸡禽杆菌aroA基因克隆及序列分析

旨在建立一种方便、高效的同源保守基因克隆方法,并对副鸡禽杆菌aroA基因进行克隆和结构分析。本试验以副鸡禽杆菌国际标准株145(C-3)基因组DNA为模板,用CODEHOP软件设计针对aroA基因的兼并引物,并运用改进的染色体步移方法扩增aroA基因序列;对该核酸序列及其编码蛋白进行结构分析并与该菌其他血清型及相关细菌进行序列比对分析。结果显示,获得了完整的aroA基因,全长1 293 bp。该基因编码由430个氨基酸组成的多肽,具有2个功能位点和5个抗原表位位点。不同血清型间氨基酸序列同源性为88.1%-100%,与其他相关细菌核酸同源性为75%以上。首次将兼并PCR和改进的染色体步移技术结合起来对副鸡禽杆菌aroA基因全长进行扩增研究,得到了预期的结果。

Molecular Cloning and Sequence Analysis of the aroA Gene of Avibacterium paragallinarum

In order to establish a convenient and efficient gene cloning method to clone unknown genes,and do some researches on molecular structure and evolutionary of Avibacterium paragallinarum(Apg) aroA complete sequence.This experiment took an international standard strain 145(C-3) of Apg as template,designed degenerate primers according to aroA gene with CODEHOP software.The improved chromosome walking was used to amplify the whole gene sequence of aroA.Some structure researches on the nucleicacid and coding protein sequence have been done.Besides,we also did some research on molecular evolution analysis with other serotypes and related bacteria by sequence alignment.Results showed the complete aroA gene of Avibacterium paragallinarum was obtained,which is 1 293 bp,and encode a protein sizes as 430 aa with two function sites and several epitopes.The homology of amino acid sequences is 88.1%-100% between different serotypes experiment strains,and the nucleic acid homology is over 75% between bacteria of the same family.This trial has combined the Degenerate PCR and improved chromosome walking for the research of genes complete sequence for the first time,and obtained the expected results.