绵羊肺炎支原体核糖体蛋白30S RPS11基因的克隆、表达及重组蛋白免疫原性研究

摘要

本研究以绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)新疆流行株基因组DNA为模板,通过PCR扩增30S RPS11基因,对其进行分子特征性分析,并将其抗原表位集中区进行原核表达,分析重组蛋白免疫原性.结果表明,30S RPS11基因全长405 bp,编码134个氨基酸,该蛋白是一种受磷酸化调控的蛋白,含有一个核糖体蛋白S11信号.SDS-PAGE结果显示,筛选的抗原表位集中区重组蛋白分子质量为29 kDa;Western blot分析显示,该重组蛋白可被MO阳性血清特异性识别,说明其具有良好的反应原性;小鼠免疫试验表明,重组蛋白诱导机体产生的间接血凝抗体效价可达1∶32~1∶64,表明其具有较强的免疫原性.本试验首次证实MO核糖体蛋白(30S RPS11)具有良好的免疫原性和反应原性,为MO血清学诊断及亚单位疫苗研发提供了科学依据.%In this study,30S RPS11 gene ofMycoplasma ovipneumoniae MO) isolated from sheep was amplified by PCR and its molecular characteristics was analyzed after sequencing.It's epitope clustering region was screened,cloned and expressed in prokaryotic cells.The results showed that the full-length of 30S RPS11 gene was 405 bp,which encoded 134 amino acids.The protein harbored a S11 signal of ribosomal protein,and could be regulated by phosphorylation.SDS-PAGE showed that the molecular mass of recombinant protein of epitope clusters was 29 kDa.Western blot analysis showed that the recombinant protein was specifically recognized by the MO positive serum,indicating that it had good reactogenicity.The immunization tests in mice showed that the recombinant protein could induce anti-serum antibody with the IHA titer of 1∶32~1∶64,which confirmed that the recombinant protein had a strong immunogenicity.In this study,the immunogenicity and reactogenicity of ribosomal protein 30S RPS11 of MO was firstly confirmed,which provided a scientific basis for the development of serological diagnostic reagents and subunit vaccine.