产毒多杀性巴氏杆菌毒素不同重组片段的免疫原性比较

Comparison of the immunogenicity of recombinant fragments of toxigenic Pasteurella multocidatoxin

为了研究多杀性巴氏杆菌毒素(PMT)片段的免疫原性,本试验对8个PMT片段分别进行了原核表达后比较分析。根据toxA基因序列,分别设计引物扩增出8个分段基因,再将其克隆到原核表达载体pET32a系统中。SDSPAGE结果表明,在大肠杆菌BL21(DE3)中均成功表达;Western-blotting结果表明,仅重组PMT1-506aa、PMT1-391aa、PMT505-1285aa和PMT973-1285aa片段的表达产物具有反应原性。将该4个重组PMT片段分别制成疫苗,皮下注射免疫小鼠,间隔14d进行二免。二免后14d,用天然PMT进行皮下注射攻毒。结果以2×LD100进行攻毒时,免疫保护效果PMT1-506aa(10/10)、PMT505-1285aa(10/10)、PMT973-1285aa(8/10)显著优于PMT1-391aa(1/10);以5×LD100进行攻毒时,免疫保护效果PMT1-506aa(10/10)、PMT505-1285aa(10/10)显著优于PMT973-1285aa(3/10)、PMT1-391aa(0/10);以10×LD100进行攻毒时,免疫保护效果PMT505-1285aa(10/10)显著优于PMT1-506aa(3/10)。本研究表明,PMT505-1285aa的免疫保护率最高,最适合作为预防PAR的候选疫苗材料。

To comparatively analyze the immunogenicity of recombinant subunit fragments of P. multocida toxin （PMT）,eight fragments were amplified from toxA by polymerase chain reaction （PCR） and then cloned into the high-expression vector pET32a. All of them were confirmed to be expressed efficiently in Escherichia coli BL21 （DE3） by SDS-PAGE. Only PMT1-5s06aa, PMT1-391aa, PMT505-1285aa and PMT973-285aa were confirmed to react specifically with the antiserum against native PMT by Western-blotting. The four fusion proteins were processed as four vaccines and were used to immunize mice subcutaneously twice at 2-week intervals, respectively. The immunized mice were subcutaneously challenged with native PMT 2 weeks after the second immunization. The pro- tection study results showed that the survival rates of PMT1-506aa （10/10）,PMT505-1285aa （10/10）and PMT973-1285aa （8/10）were significantly higher than that of PMT1-391aa （1/10）when challenged with 2 × LD100 of native PMT, the survival rates of PMT1-506aa （10/10） and PMT505-1285 （10/10） were significantly higher than that of PMT973-1285aa （3/10）and PMT1-391aa （0/10）when challenged with 5 × LDloo toxin,the survival rate of PMT505-1285aa （10/10）was significantly higher than that of P MT1-506aa （3/ 10）when challenged with 10 × LDloo toxin. Therefore, PMT505-1285aa with the highest survival rate was successfully screened to be an attractive candidate for a subunit vaccihe against PAR.