重组腐败梭菌α毒素对兔的免疫效力分析  

Evaluation of Protective Efficacy of Recombinant Clostridium septicum α Toxin in Rabbits

作　　者：陈小云[1] 刘莹[1] 薛麒[1] 朱真[1] 李启红[1] 印春生[1] 姚文生[1] 康凯[1] 杜吉革 CHEN Xiao-yun;LIU Ying;XUE Qi;ZHU Zhen;LI Qi-hong;YIN Chun-sheng;YAO Wen-sheng;KANG Kai;DU Ji-ge(China Institute of Veterinary Drug Control,Beijing 100081,China)

机构地区：[1]中国兽医药品监察所,北京100081

出　　处：《畜牧兽医学报》2018年第8期1727-1734,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：科技部十三五"牛羊重要疫病免疫防控新技术研究"重点专项子课题(2017WFD0500903);中国兽医药品监察所所级课题(201702)

摘　　要：本研究旨在获得重组腐败梭菌α毒素,并评价其毒力及免疫原性。对已知的腐败梭菌α毒素编码基因进行优化设计,通过人工合成获得基因片段Gcsa。将Gcsa克隆至原核表达载体pET-30a(+)中进行表达与纯化,从而获得重组蛋白rCSA。利用Western blot方法检测纯化的rCSA与腐败梭菌α毒素抗血清的反应性,并采用犬肾细胞系(MDCK)和小鼠来检测rCSA的毒力。以甲醛脱毒后的rCSA与Montanide ISA 201佐剂以1∶1的比例制备疫苗,免疫家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测兔血清的中和抗体效价。结果表明,rCSA为可溶性表达,比例可达50%,且能与腐败梭菌抗血清反应。MDCK细胞毒性试验显示,0.15ng·mL^(-1)的rCSA即可引起明显的细胞病变;小鼠安全性试验显示,rCSA对ICR小鼠的最小致死量是1.5×10~6 ng·kg^(-1);每毫升的一免抗血清可中和100~130个小鼠MLD的腐败梭菌毒素;每毫升的二免抗血清可中和360~480个小鼠MLD的腐败梭菌毒素;1个家兔MLD的腐败梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔得到了4/4的保护。以上结果表明,rCSA具有良好的免疫原性,可作为腐败梭菌病基因工程亚单位疫苗的候选抗原。This study was conducted to obtain the recombinant Clostridium septicumαtoxin and to evaluate its virulence and immunogenicity.Based on the known sequence,Gcsa,αtoxin gene of C.septicum,was optimized and synthesized.Then,Gcsa was cloned into prokaryotic expression vector pET-30 a（＋）for expression and purification.Reactivity of the purified protein（rCSA）with antiserum of C.septicum was detected by Western blot.Meanwhile,Canine Kidney（MDCK）cell and mice were used to detect the virulence of rCSA.According to the method prescribed in Chinese Veterinary Pharmacopoeia（2015）,four rabbits were immunized with rCSA emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer.The results showed that rCSA was expressed at a high level as soluble form with a ratio about 50% by gray scale scanning,and it could react with the antiserum of C.septicum.For MDCK cell,the concentration of rCSA to induce apparent cytopathic effect（CPE）was found to be 0.15 ng·mL^-1.At the same time,the Minimum Lethal Dose（MLD）of rCSA to ICR mice has been calculated as 1.5×10^6 ng·kg^-1.After the first immunization,sera from rabbits immunized with rCSA couldneutralize 100^-130 mice MLD C.septicumtoxin per milliliter,and 360-480 mice MLD after twice immunization.Moreover,rabbits in rCSA immunized group fully survived from the challenge of C.septicumtoxin at the dose of 1 rabbit MLD,whereas all of the rabbits died（4/4）in the control groups.These data suggest that rCSA has good immunogenicity,and can be used as a potential vaccine candidate for genetic engineering subunit vaccine of C.septicum.

关 键 词：腐败梭菌α毒素 重组表达 毒力 抗原性 

分 类 号：S855.1[农业科学—临床兽医学]