截短腐败梭菌α毒素的表达及其免疫原性评价

Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum

为了获得截短腐败梭菌α毒素并评价其毒性、反应原性和免疫原性,根据腐败梭菌α毒素的结构与功能,设计2对引物分别扩增含有穿孔活性区和受体结合区的α毒素基因csaN和csaC,克隆至pET-28a(+)质粒,构建截短α毒素表达载体,转化大肠杆菌BL21(DE3),诱导csaN和csaC表达,鉴定产物的反应原性和毒性,将经过诱导表达的工程菌灭活制成铝佐剂疫苗免疫家兔1次,通过血清中和试验和攻毒保护试验评价免疫效力,设立重组α全毒素rcsa免疫组作为阳性对照。结果显示,2个截短α毒素在大肠杆菌BL21(DE3)中均获得表达,产物能够与腐败梭菌抗毒素血清发生特异性反应,但失去了小鼠致死毒性。免疫家兔血清中和试验结果显示,csaN组血清的中和效价为1 MLD/0.1 mL,达到《中华人民共和国兽药典三部(2015年版)》效力检验的最低标准,csaC组为0,rcsa组为8 MLD/0.1 mL;csaN和csaC组家兔用1 MLD毒素攻击均未获得保护,而rcsa组家兔4/4获得保护。结果表明,截短α毒素与全毒素相比,失去了毒性但保留了反应原性,仅含穿孔活性结构区的截短部分具有较弱的免疫原性。

In order to acquire truncated alpha toxins of Clostridium septicum and evaluate their toxicity,reactionogenicity and immunogenicity,two pairs of primers were designed to amplify csa N and csa C genes,which contained a pore-forming activity gene site and a binding cell receptor gene site,respectively.The two genes were cloned into the plasmid vector p ET-28a(+),and the recombinant plasmids were transformed into Escherichia coli BL21(DE3). The toxicity and reactionogenicity of the two genes induced products were identified. After induction and inactivation,the two gene-induced products were mixed with an adjuvant aluminum hydroxide solution and prepared into vaccine. Rabbits were immunized once with the vaccine. The immune effect was evaluated by toxin challenge and serum neutralization tests with the recombinant complete alpha toxin vaccine as a positive control. The results showed that two truncated alpha toxins were expressed effectively,and could react with the antiserum of C.septicum,but did not kill the mice. The immune serum neutralization titers of the rabbits in the csa N and rcsa group were 1 and 8 MLD/0.1 mL in lining with the parameters stipulated in the Chinese Veterinary Pharmacopoeia:Volume 3(2015),but one of the csa C group was 0 MLD/0.1 mL.The rabbits in thecsa N,csa C and negative control groups died when challenged with 1 MLD of C.septicum toxin,whereas 4/4rabbits in the rcsa group survived.The results suggested that the two truncated alpha toxins had reactionogenicity but no toxicity,the truncated alpha toxin containing the pore-forming activity site had limited immunogenicity compared to the recombinant complete alpha toxin.