猪产毒多杀性巴氏杆菌皮肤坏死毒素的细胞毒性及其抗体的制备

Characterization and Its Antibody Preparation of Pasteurella multocida Toxin

将猪产毒多杀性巴氏杆菌(toxigenicPasteurella multocida,T+Pm)增菌后经超声波破碎,用DEAE-52阴离子交换柱和Sephadex G-200处理,提纯天然多杀性巴氏杆菌皮肤坏死毒素(Pasteurella multocidatoxin,PMT)。经Western blot和细胞毒性试验证实,该毒素具有良好的反应原性,且7 ng/mL的PMT能使Vero细胞病变。用该毒素制成的类毒素菌苗免疫2头健康断奶仔猪,同时设T+Pm灭活苗和PBS对照组。4次免疫后,琼脂扩散试验检测类毒素菌苗组毒素抗体效价达1∶16,而灭活苗组未见毒素抗体产生。用200μg提纯的PMT和50亿T+Pm混合液经耳静脉攻毒各组试验猪,结果发现对照组和灭活苗组试验猪在攻毒后出现明显的急性败血症状,未产生毒素抗体;类毒素菌苗组试验猪10 d后耐过,经颈动脉采血收集高免血清,效价达1∶32。

PMT was purified from sonic extracts of a serotype D strain of toxigenic Pasteurella multocida（T＋Pm）,with a DEAE-52 sephacel column and a sephadex G-200 column.Western blot confirmed that the toxin could specifically react with antiserum against T＋Pm.Cytotoxicity on Vero cells showed that there were pathologically changes after administration of 7 ng/mL purified toxin.The toxin was emulsified with Freund＇s adjuvant in equal volumes to get toxoid vaccine.Groups of healthy pigs were vaccinated four times with an interval of two weeks.The neutralization antibody titer of toxoid vaccine detected by the agar gel diffusion test was 1∶16.However,groups of inactivated bacterin and negative control didn＇t produce antibodies against the toxin.All pigs were challenged intravenously with compounds of 200 μg toxin and 5 × 109 cfu HN-13 strain of T＋Pm.The protection efficiency of toxoid vaccine group was 100%,and the neutralization antibody titer was 1∶32.Inactivated bacterin and negative control groups couldn＇t provide any protection.The data revealed that the toxin had immunogenicity and the prepared antiserum could be used to develop diagnosis methods.