绵羊肺炎支原体P110/LppT黏附素家族保守区的原核表达和免疫原性分析

Prokaryotic Expression and Immunogenicity Analysis of Conserved Region of P110/LppT Adhesin Family in Mycoplasma ovipneumoniae

绵羊肺炎支原体(Mo) NM2010株的P120蛋白与猪肺炎支原体(Mhp)的黏附素P110高度同源,同属于P110/LppT黏附素N端结构域家族。为了筛选用于研制广谱绵羊支原体肺炎疫苗的保护性抗原,本试验利用生物信息学方法对Mo NM2010株P120蛋白的保守区段、结构、B细胞抗原表位进行预测分析,并对P120基因的N端和C端2个保守区基因片段进行克隆、表达、可溶性分析和抗原特性分析;2个表达产物分别对兔进行免疫试验,用ELISA方法检测免疫兔血清抗体效价和IL-2、IL-4、IL-10和IFN-γ细胞因子浓度。结果显示,P120蛋白N端有信号肽和1个跨膜螺旋,亚细胞定位在外膜,可能是一种外膜蛋白,P120蛋白N端和C端2个保守区段均有较多B细胞线性抗原表位,具有较好的抗原性;经序列测定证实,合成的P120-N和P120-C 2个基因片段的基因序列完全正确,长度分别为1 509 bp和741 bp,表达的重组蛋白分子质量分别为58.1 kDa和29.5 kDa;P120蛋白N端以可溶和包涵体2种形式存在,C端以包涵体形式存在,均具有良好的抗原性;新西兰大白兔经4次免疫后,重组蛋白P120-N组和重组蛋白P120-C组兔血清中均产生了特异性抗体IgG,效价分别为1∶512 000和1∶128 000;与阴性对照组相比,重组蛋白P120-N组和重组蛋白P120-C组兔血清中IL-2、IL-4、IL-10和IFN-γ浓度均极显著升高(P<0.01)。结果表明,重组蛋白P120-N和P120-C可以诱导试验动物机体的体液免疫反应,P110/LppT黏附素家族保守区可以作为广谱绵羊支原体肺炎疫苗的候选蛋白。

The P120 protein of Mycoplasma ovipneumoniae(Mo) NM2010 strain is highly homologous with the adhesin P110 of Mycoplasma hyopneumoniae(Mhp),and both belongs to the N-terminal domain family of P110/LppT.In order to screen for protective antigens for the development of a broad-spectrum vaccine against mycoplasmal pneumonia of sheep,this study used bioinformatics methods to predict and analyze the conserved regions,structure,and B cell antigenic epitopes of the P120 protein of the Mo NM2010 strain.And two conserved gene fragments from the N-terminus and C-terminus of P120 were cloned,expressed,and analyzed for solubility and antigenic properties.Two expressed products were used to immunize rabbits respectively,and the serum antibody titers and cytokine concentrations of IL-2,IL-4,IL-10 and IFN-γ were detected by ELISA.The results showed that the N-terminal of the P120 protein contained a signal peptide and a transmembrane helix,with subcellular localization in the outer membrane,suggesting it may be an outer membrane protein.Both the N-terminal and C-terminal conserved regions of P120 had multiple linear B cells antigenic epitopes,indicating good antigenicity.The results of sequence analysis confirmed the complete and accurate gene sequences of the synthesized P120-N and P120-C gene fragments,with lengths of 1 509 bp and 741 bp,respectively,and the expressed recombinant proteins had molecular masses of 58.1 kDa and 29.5 kDa,respectively.The N-terminal of P120 existed both in the forms of soluble and inclusion bodies,while the C-terminal existed in the form of inclusion bodies,both showing good antigenicity.After four immunizations,specific IgG antibodies were generated in the sera of New Zealand white rabbits in both recombinant protein P120-N group and recombinant protein P120-C group,with Ig G antibody titers of 1∶ 512 000 and 1∶ 128 000,respectively.Compared with the negative control group,the concentrations of IL-2,IL-4,IL-10 and IFN-γ in sera of rabbits in both the recombinant protein P120-N group and recombinant protein P120-C group significantly increased( P<0.01).The results indicate that the recombinant proteins P120-N and P120-C can induce humoral responses in experimental animals,indicating that the conserved region of P110/Lpp T adhesin family can be used as a candidate protein for broad-spectrum vaccine against mycoplasmal pneumonia of sheep.