C型产气荚膜梭菌产α毒素能力分析及免疫原性试验研究

Analysis of Alpha-toxin Production Ability and Immunogenicity Test of C-type Clostridium Perfringens

利用16S rRNA分子鉴定技术对病死羊体内分离的19株野毒株产气荚膜梭菌进行了分子生物学鉴定,从中筛选出产毒素稳定的一株C型产气荚膜梭菌(海C1株),并通过优化培养基配方及培养方式提高其产毒素能力,最终利用该菌株研制了羊猝疽灭活疫苗,完成了疫苗对家兔及羊的效力试验。结果显示,该菌株与现用羊瘁狙疫苗生产株分子系统发育树亲缘关系较近;该菌株(海C1株)在含1.0%葡萄糖,pH 8.4~8.6的VF培养基中36℃3~6h产毒最高可达7000 MLD/mL;利用该菌株研制的灭活菌苗对家兔的免疫保护力是1~1.5 MLD/mL(家兔静注),对绵羊的免疫保护力是4~16 MLD/mL(绵羊静注)。免疫羊血清抗体效价达8~12 MLD/mL(小鼠静注)。该菌株具有较强的产毒稳定性和较好的免疫原性,可作为羊猝疽疫苗生产备选菌株。

the molecular identification of 19 wild-type Clostridium perfringens isolated from dead sheep was carried out by using 16S rRNA molecular identification technology,and a stable toxin-producing strain of C-type Clostridium perfringens(sea C1 strain)was selected.its toxin production ability were improved by optimizing the medium formula and culture method,and finally using this strain to develop a goat gangrene inactivated vaccine,and then the vaccine's efficacy test was completed in rabbits and sheep.The results show that this strain is closely related to the molecular phylogenetic tree with the current sheep sniper vaccine production strain;this strain(sea C1 strain)was cultured under containing 1.0%glucose,pH 8.4 to 8.6,at 36°C 3h-6h,toxin poduction can reach up to 7000 MLD/mL;the immune protection of the inactivated vaccine developed using this strain is 1-1.5 MLD/mL(rabbit intravenous injection),and the immune protection of sheep is 4-16MLD/mL(sheep intravenous injection).Immune goat serum antibody titer reaches 8-12MLD/mL(mouse intravenous injection).This strain has strong toxin production stability and good immunogenicity,and can be used as a candidate for the production of sudden gangrene vaccine Strains.