鼠疫耶尔森菌Pla基因的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of Pla gene in Yersinia pestis

鼠疫(plague)是一种重要的人兽共患病,给人类健康带来严重的威胁。本研究为预测Pla(plasminogen activator)基因的结构、功能及作为优势抗原的可能性,提取鼠疫耶尔森菌的基因组DNA进行目的基因扩增,并构建pMD19T-Pla克隆载体,利用生物信息学软件对该基因编码蛋白的理化性质、亲/疏水性、跨膜区、信号肽、磷酸化位点、糖基化位点、保守结构域、二级结构、三级结构以及T/B细胞抗原表位进行预测和分析。结果显示,Pla基因具有完整的ORF,编码312个氨基酸,Pla蛋白理论等电点为5.88,原子组成为C_(1538)H_(2307)N_(415)O_(486)S_(7),属不含跨膜区亲水的稳定蛋白,在第1~23氨基酸位点含有信号肽,共含48个磷酸化位点,其中丝氨酸位点含量最高。二级结构预测显示,主要由延伸链和无规则卷曲组成,分别占28.21%和47.44%。抗原表位预测显示,该蛋白含有较多的B细胞抗原表位,分别位于29~35、43~50、54~61、96~125、127~136、141~149、177~182、184~230、233~244、266~302位氨基酸附近。结果表明,Pla蛋白具有较好的生物学特性及抗原性,为鼠疫耶尔森菌新型疫苗的研制及候选抗原的筛选奠定基础。

Plague is an important zoonotic disease that poses a serious threat to human health.In this experiment,in order to predict the structure and function of the plasminogen activator(Pla)gene and the possibility of being a dominant antigen,the genomic DNA of Yersinia pestis was extracted for the purpose of gene amplification,and the pMD19T-Pla cloning vector was constructed.Using bioinformatics software for the physical and chemical properties of the gene,affinity/hydrophilicity,transmembrane region,signal peptide,phosphorylation site,glycosylation site,conserved domain,secondary structure,tertiary structure,and B/T cell epitopes are predicted and analyzed.The results show that the Pla gene has a complete ORF,encoding 312 amino acids,Pla protein has a theoretical isoelectric point of 5.88 and an atomic composition of C_(1538)H_(2307)N_(415)O_(486)S_(7).It is a stable protein that does not contain a transmembrane region and has a signal peptide at the 1st to 23th amino acid positions.The hydrophilic protein contains a total of 48 phosphorylation sites,of which the serine phosphorylation site is the highest.The secondary structure is mainly composed of extended chains and random curls,accounting for 28.21%and 47.44%,respectively.Antigen epitope prediction shows that the protein contains more B-cell epitopes,which are located at 29~35,43~50,54~61,96~125,127~136,141~149,177~182,184~230,233~244,266~302 amino acids.Analysis shows that the Pla protein has good biological characteristics and antigenicity.This study has laid a solid foundation for the development of new Yersinia pestis vaccine and screening of candidate antigens.