摘要
【方法】利用PCR方法对绵羊肺炎支原体(Mycoplasma ovipneumoniae,MO)新疆分离株MO-XJ p74基因进行扩增、克隆及测序,分析其分子特征。将P74蛋白抗原集中区编码序列亚克隆至表达载体p ET-32a(+),构建重组表达载体p ET-32a(+)-p74C,转化至E.coli BL21(DE3)感受态细胞中,IPTG诱导表达。【结果】MO-XJ p74基因全长2016 bp,编码671个氨基酸;对编码的氨基酸序列分析发现,该蛋白不含信号肽序列,9~31位氨基酸序列含有1个跨膜区,有16个N-糖基化位点、7个N-酰基化位点、17个酪蛋白激酶Ⅱ磷酸化位点及5个蛋白激酶C磷酸化位点。同源性分析显示MO-XJ P74蛋白氨基酸序列与标准株MO-SC01氨基酸序列同源率为86.5%,其羧基端为P74蛋白抗原集中区。SDS-PAGE检测结果显示,表达的P74蛋白抗原表位集中区片段对分子质量约为35.5 k Da,与理论值相符;Western blot分析表明重组P74蛋白具有较强的反应原性。【结论】本研究为进一步筛选MO亚单位疫苗及血清学诊断的候选抗原奠定了前期基础。
[ Method ] The p74 gene of Mycoplasma Ovipneumoniae （MO） Xinjiang isolate （MO-XJ） was amplified by PCR and then cloned, sequenced. The molecular biological characteristics were analyzed and the main antigenic domain of the P47 protein were predicted. The am- plified products were then further subcloned in the expression vector pET-32a（ ＋ ） to obtain the recombinant expression pET-32a（ ＋ ） -p74C vector. The recombinant expression pET-32a（ ＋ ） -p74C vector was transformed into E. coli competent cells of BI2.1 （ DE3 ） and induced by IPTG. [ Result] Bioinformatics analysis showed that the p74 gene had a length of 2016 bp and encoded 671 aa. The results revealed that the p74 protein had no signal peptide sequence and 1 transmembrane domain sequence from 9 to 31 amino acid residues, with 16 N-glycosylation sites, 7 N-acylated sites 17 casein kinase 11 phosphorylation sites and 5 protein kinase C phosphorylation site. P74 protein of MO-XJ shared 86.5 % identities with the standard strain MO-SCO1. The advantage B-cell antigenic domain exists in the carboxyl terminal. SDS-PAGE showed that recombinant protein was successfully expressed with a mass of molecule of 35.5 kDa. Western blot revealed that the recombinant P74C protein could specifically react with anti-MO positive serum. [ Conclusion]This study lays foundation for further screening of candidate antigens for subunit vaccine MO and detection of serological diagnosis.
出处
《西南农业学报》
CSCD
北大核心
2017年第5期1222-1228,共7页
Southwest China Journal of Agricultural Sciences
基金
国家自然科学基金(31360596)
国家国际科技合作专项(2014DFR31310)
关键词
绵羊肺炎支原体
p74基因
克隆
序列分析
原核表达
反应原性
Mycoplasma ovipneumoniae
p74 gene
Cloning
Sequence analysis
Prokaryotic expression
Reactogenicity
