肺炎支原体P1黏附蛋白的表达及初步应用研究

Expression and Preliminary Application of the P1 Adhesion Protein of Mycoplasma Pneumoniae

目的对肺炎支原体(Mycoplasma pneumoniae,Mp)3′端的P1黏附蛋白基因片段进行密码子优化与重组表达、蛋白纯化、表达产物的免疫性分析及临床诊断初步应用研究。方法选择MpFH株P1蛋白序列优化并合成933bp的3′端p1反义基因片段(p1′),将此基因片段插入表达载体pET-GST后,转入大肠杆菌BL21。诱导阳性克隆株表达重组蛋白后,纯化该蛋白质,用肺炎支原体感染阳性患者,血清通过免疫印迹实验鉴定其免疫反应性。结果诱导表达的重组蛋白经SDS-PAGE分析,重组蛋白的相对分子量约为59kDa。免疫印迹实验证实重组蛋白P1可与阳性肺炎支原体感染患儿血清发生特异性反应,用P1蛋白建立的间接ELISA法,其敏感性和特异性分别为95.5%和95.45%。结论已成功地在大肠杆菌中表达了P1蛋白,为MP感染的快速诊断提供了一种新的方法,也为进一步探讨P1蛋白的功能及其在MP感染发病机制中的作用奠定了基础。

Objective The 3＇terminal end of the P1 adhesin gene fragment of Mycoplasma pneumoniae （Mp） was cloned, expressed and purified, and its immune reactivity of the expressed products was analyzed in the present investigation, in order to provide the basis for diagnosis development. Methods In this study, the 3＇end of the P1 gene fragment （p1＇） in FH strain of Mp was used in codon optimization with information biology methods and inserted into the vector pET-GST. The recombinant plasmid was used to transform competent E. coli strain BL21 by IPTG induction. Western blotting assay was used to determine the immunoreactivity of the purified recombinant proteins. Results The experimental results showed that recombinant protein with molecular weight of 59 kDa was found to be expressed which was demonstrated by SDS-PAGE analysis, and this protein was proved to be P1 adhesin antigen, as analyzed by Western blotting. It showed specific reaction of P1 protein with the sera of children with MP infection. The sensitivity, specificity of LISA developed using P1 as an antigen were 95. 5% , 95. 45%, respectively. Conclusion Recombinant P1 protein was successfully expressed in E. coli, which provided a novel tool for rapid diagnosis of MP infection and laid a foundation of further study on function of P1 as well as the role of P1 in onset mechanism of MP infection.