猪链球菌LPxTG蛋白HP0197与外源蛋白的融合表达及融合蛋白抗原活性的检测

Fusion expression of Streptococcus suis LPxTG protein HP0197with exogenous protein and evaluation of its antigenicity

为了从蛋白水平探究猪链球菌(S. suis)的表面LPxTG(Leu-Pro-x-Thr-Gly,x表示任何氨基酸)蛋白HP0197在S. suis表面展示外源抗原的可行性,本研究分别经PCR扩增HP0197蛋白的功能区编码基因片段hp0197(去除了HP1097全长蛋白aa1~aa40的转运信号肽序列和aa524~aa56的CWA基序)、hp0197-18 ku基因片段(即HP0197蛋白的18 ku结构域的编码基因)、hp0197-Loop基因片段(HP0197蛋白aa201~aa523的编码基因片段),并通过融合PCR将gfp基因融合至上述两段基因即HP0197蛋白的18 ku结构域和Loop区编码基因之间,并构建表达HP-GFP融合蛋白的重组质粒及表达HP0197蛋白的重组质粒,经测序鉴定后将其分别转化大肠杆菌BL21(DE3),并经IPTG诱导后采用SDS-PAGE鉴定。采用Ni-NTA亲和树脂方法纯化HP0197蛋白和HP-GFP融合蛋白后分别免疫小鼠,经间接ELISA检测首免后不同时间各组小鼠血清中的HP0197抗体和GFP抗体水平,评价HPGFP融合蛋白和HP0197蛋白的抗原活性。SDS-PAGE结果显示,分别经原核系统表达并纯化了HP-GFP融合蛋白和HP0197蛋白。间接ELISA结果显示,单独免疫HP0197蛋白的小鼠产生了HP0197抗体,但未产生GFP抗体;而免疫HP-GFP融合蛋白的小鼠,既产生了HP0197抗体,也产生了GFP抗体,且在首免后28 d这两种抗体效价均达1:204 800。上述结果表明在HP0197蛋白的18 ku结构域和Loop区之间插入外源蛋白,可在保留HP0197蛋白自身抗原活性的前提下,有效发挥外源蛋白的抗原活性。本研究首次在蛋白水平上证实了S. suis的LPxTG蛋白HP0197作为载体蛋白用于在S. suis表面展示外源蛋白的可行性,为进一步构建S. suis表面蛋白展示系统奠定了基础。

To explore the feasibility of using the LPxTG(Leu-Pro-x-Thr-Gly, x means any amino acid) protein HP0197 to display exogenous antigen on the surface of Streptococcus suis at protein level, the functional region coding gene fragment hp0197(the transport signal peptide sequence of aa1-aa40 and the CWA motif of aa524-aa56 of HP1097 full-length protein were removed),the fragment hp0197-18ku(encoding the 18ku domain of HP0197 protein) and the fragment hp0197-Loop(encoding amino acids aa201-aa523 of HP0197 protein) were amplified by PCR, respectively, and the gfp gene was fused between the 18 ku domain and Loop region coding fragments of HP0197 protein by fusion PCR. The recombinant plasmid expressing HP-GFP fusion protein and the recombinant plasmid expressing HP0197 protein were constructed. After sequencing and identification, each was transformed into E. coli BL21(DE3), and induced by IPTG and identified by SDS-PAGE. After purification by Ni-NTA affinity resin, the HP0197 protein and HP-GFP fusion protein were used to immunize mice, respectively. The levels of HP0197 antibody and GFP antibody in the serum of mice in each group at different time-points after the first immunization were detected by indirect ELISA so as to evaluate the antigenicity of HP-GFP fusion protein and HP0197 protein. The results of SDS-PAGE showed that the HP-GFP fusion protein and HP0197 protein were expressed and purified by prokaryotic system, respectively. The ELISA results showed that the mice immunized with HP0197 protein produced only HP0197 antibody, and no GFP antibody. In contrast, the HP-GFP fusion protein immunized mice produced both HP0197 and GFP antibodies, and the antibody levels reached 1:204800 at 28 th day post the first vaccination. These results indicate that inserting exogenous protein between the 18 ku domain and Loop region of HP0197 protein can effectively exert the antigenicity of exogenous protein while retaining the antigenicity of HP0197, confirming the feasibility of the HP0197 protein of S. suis as a carrier protein to display exogenous protein on the surface of S. suis at the protein level. This study laid the foundation for the construction of the protein display system on surface of S. suis.