产气荚膜梭菌多毒素融合蛋白的表达与纯化及基因工程亚单位多价疫苗的制备

Expression and purification of multiple toxin fusion protein and preparation of gene engineering subunit vaccine of Clostridium perfringens

产气荚膜梭菌(Clostridium perfringens)的α、β2和ε毒素是病原的主要外毒素,也是制备预防该病基因工程亚单位多价疫苗的主要研究对象。本研究通过优化密码子、去除蛋白信号肽、选择亲水性与抗原性较好的序列、同时优化表达条件等方法的探索,在大肠杆菌表达系统中获得了高效表达的可溶性α-β2-ε融合蛋白;用该蛋白免疫小鼠后可产生较高水平的血清抗体,针对A、B、C和D型产气荚膜梭菌的免疫保护率分别为100%,100%,90%,100%;小鼠三免后7~14d的抗体效价达到峰值。本研究构建了多毒素融合蛋白表达载体Pet30a-α-β2-ε,并成功表达与纯化出了高效可溶性的目的蛋白,且表达出的融合蛋白具有良好的免疫原性,可以进一步用于羊三联四防基因工程亚单位疫苗的研制,具有较强的研究价值和应用前景。

Alpha, beta 2 and epsilon toxin of the pathogen are the main exotoxin in Clostridium perfringens,and they are main research direction of the gene engineering subunit multivalent vaccin. According to optimizing codon,removing the signal peptide, selecting better hydrophilicity and antigenic sequences ,and optimizing the expression conditions in this study, we obtained the soluble alpha-beta 2-epsilon fusion protein in Escherichia coli expression system. In addition,on the basis of alpha-beta 2-epsilon fusion protein,we developed genetic engineering subunit vaccine for inhibiting mixed infection of several types Clostridium perfringens. The serum antibody level rose obviously in the anminal immuned with the vaccine. The vaccine can resist the attack of type A,B,C and D of Clostridiurn perfringens, and immune protection rate of the mouse were 100%, 100%, 90% and 100% ,respectively. At 7 to 14 days after the third immune,the antibody titer reached a peak. This research constructed the multiple toxin fusion protein expression vector Pet30a-α-β2-ε and successfully expressed and purified the soluble target protein. The soluble fusion protein had good immunogenicity,could be used as a vaccine to control the disease caused by Clostridium perfringens infection,and had good research value and application prospect.