构象稳定的破伤风毒素Hc 片段突变体 (HcM) 的制备及其免疫原性分析

Production and immunogenicity analysis of conformation-stable fragment-C mutant of tetanus toxin

摘要:

破伤风是由破伤风杆菌侵入人体伤口、生长繁殖、产生毒素而引起的一种急性特异性感染，其死亡率高，严重危害人民生命健康。研究证实破伤风毒素重链C 端 (Hc) 具有与毒素受体结合的活性，完全保留了全分子的免疫原性，有望开发成为新的基因工程破伤风亚单位疫苗以替换传统的甲醛灭活类毒素疫苗。由于野生型Hc 蛋白 (HcW) 易形成分子间及分子内二硫键，且各构象分子之间易发生不稳定的转换，为疫苗的生产工艺带来困难，因此，通过将破伤风HcW 蛋白的869 位半胱氨酸突变为丙氨酸，构建构象稳定的破伤风亚单位疫苗突变体HcM

Abstract:

Tetanus is caused by tetanus toxin synthesized by Clostridium tetani. Fragment C (Hc), the 50 kDa carboxy-terminal portion of tetanus toxin, is nontoxic but has receptor protein binding activities, which has been evaluated as a potential new recombinant subunit vaccine to replace the traditional formaldehyde inactivated toxoid vaccine. It is easy for wild Hc (HcW) to form inter- and intra-molecular disulfide bonds and the different conformations changes unstably, which brings difficulties for vaccine production technology. In our study, the Cys 869 of HcW was mutated to Ala and the conformation-stable fragment-C mutant of tetanus toxin (HcM) was constructed. The HcM was expressed, fermented and purified and its stability, receptor binding and immunogenicity were evaluated. The result showed that the HcM got high-level expression and was purified to >95% of purity. The purified HcM was conformation-stable at different temperature for different time and kept the binding activities with one of its receptor GT1b. Mice given three vaccinations by HcM developed a protective immune response and were 100% protected against an intraperitoneal administration of 1×10³ 50% lethal doses (LD₅₀s) of tetanus neurotoxin. All the results showed that the conformation-stable HcM had potent immunogenicity as a recombinant tetanus vaccine candidate with simple production process and similar immunogenicity with HcW. Whether for routine tetanus therapy or for countries to respond to unexpected events (war, earthquake or other disaster), it is of great significance.