腐败梭菌无毒重组α毒素的表达与免疫保护性分析

本研究旨在获得腐败梭菌无毒重组α毒素，并评价其免疫保护性。对已知的腐败梭菌α毒素编码基因进行优化设计和人工合成，获得了缺少第212-222位共11个氨基酸编码序列的基因片段（GCSA_Δ11）。将该基因克隆至原核表达载体pET-30a（+）中进行表达与纯化。利用Western blot方法检测获得的重组α毒素（rCSA_Δ11）与腐败梭菌天然毒素抗血清的反应性，并采用小鼠检测其毒力。随后，以rCSA_Δ11制备疫苗免疫家兔，按照《中华人民共和国兽药典》（2015年版）规定的方法检测家兔血清的中和抗体效价。结果表明，rCSA_Δ11可溶表达比例可达46%，且能与腐败梭菌天然毒素抗血清反应。进一步的试验结果显示，rCSA_Δ11丧失了对小鼠的毒力，0.1 mL的一免兔血清可中和8~12个小鼠最小致死量（MLD）的腐败梭菌天然毒素；二免兔血清可中和32~45个小鼠MLD的腐败梭菌天然毒素。1个家兔MLD的腐败梭菌天然毒素攻毒后，对照组家兔4/4死亡，免疫组家兔得到了100%（4/4）的保护。综上表明，无毒性的rCSA_Δ11保留了良好的免疫保护性，从而为腐败梭菌病基因工程亚单位疫苗的研制提供了重要的试验数据。

Expression and Immunogenicity of No-toxin Recombinant Clostridium septicum α Toxin

This experiment was conducted to express recombinant Clostridium septicum α toxin without toxin and subsequently evaluate the immunogenicity of the recombinant toxin. Recombinant α toxin gene (GCSA_Δ11) of Clostridium septicum deleted 11 amino acids (212-222) was synthesized based on the sequence reported. Subsequently, GCSA_Δ11 was cloned into the prokaryotic expression vector pET-30a (+) to get the recombinant protein (rCSA_Δ11). The reactivity of rCSA_Δ11 with antiserum of Clostridium septicum crude toxin was detected by Western blot, and mice were used to detect the virulence of rCSA_Δ11. The rabbit antiserum against the recombinant protein was prepared and the neutralizing titer was detected according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein rCSA_Δ11 was expressed at a high level as soluble form with a ratio about 46%, and the protein without virulence to mouse could react with the antiserum of Clostridium septicum toxin; rabbit antiserum after first immunization could neutralize 8-12 mice MLD of Clostridium septicum crude toxin per 0.1 mL, and 32-45 MLD after twice immunizations. After challenge with 1 MLD of Clostridium septicum crude toxin, the rabbits immunized with rCSA_Δ11 were protected with a ration of 100%, whereas all of the rabbits died (4/4) in the control groups. The results suggest that the recombinant of Clostridium septicum α toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of Clostridium septicum genetic engineering subunit vaccine.