重组产毒多杀性巴氏杆菌毒素PMT的N-端和C-端蛋白 的生物学活性及免疫原性

将5个toxA基因片段N1518、C2345、N3172、N3388和C2115分别克隆到合适的原核表达载体pET-28a(b,c)系统,其中pET28a-N1518和pET28b-C2115在大肠杆菌成功表达,获得大小分别为57kDa和78kDa的融合蛋白rPMT-N和rPMT-C,Western blot检测证实两种表达产物均具有反应原性.分别以200μg rPMT-N和rPMT-C对小白鼠进行体内生物学活性试验,结果两种表达蛋白均不能致死小白鼠;体外细胞毒性试验证实896ng/mL的rPMT-N能使Veto细胞发生病变,而rPMT-C对Veto细胞无明显毒性作用.将rPMT-N和rPMT-C制成亚单位疫苗,同时设天然PMT及无菌PBS对照组,间隔2周分2次皮下免疫小白鼠.二免后2周用8.2×105 CFU的HN-13株T Pm进行腹腔攻毒,结果rPMT-N组保护率为90.0%(9/10),rPMT-C组保护率为50.0%(5/10),天然PMT组保护率为80.0%(8/10).综上试验表明,rPMT-N具有良好的生物学活性和免疫原性,可作为PAR疫苗添加成分,显示了良好的应用前景.

Characteristics and immunogenicity of the N-terminal and C-terminal re-combinants of Pasteurella multocida toxin

In this study, five fragments of recombinant subunit Pasteurella multocida toxin (PMT) were constructed. Only pET28a-N1518 and pET28b-C2115 could be expressed efficiently in Escherichia coli. The molecular weight of the fusion proteins was 57 kDa and 78 kDa .Western blot confirmed that the two proteins could specifically react with antiserum against Pasteurella multocida toxin. No mice died after the intraperitoneal administration of these two proteins with the dose of 200 microg per mouse, but Vero cell was pathologically changed after administration of 896ng/mL rPMT-N. The fusion protein of rPMT-N and rPMT-C was purified, and emulsified with Freund's adjuvant in equal volumes to get subunit vaccine. Groups of Kunming mice were immunized subcutaneously twice at interval of two weeks. All mice were challenged intraperitoneally with 8.2 x 10(5) CFU HN-13 strain of T+Pm. The protection efficiency of rPMT-N, rPMT-C and crude PMT against HN-13 strain were 90%, 50% and 80%, respectively. The data revealed that the fusion protein of rPMT-N had immunogenicity and potential for developing a subunit vaccine against PAR in pigs.