产气荚膜梭菌ε毒素突变体的表达及免疫保护力评价

Expression and Evaluation of Protective Efficacy of Clostridium perfringens ε Toxin Mutant

为获得产气荚膜梭菌ε毒素(ETX)的重组突变体,并评价其毒力及免疫原性。对已知的D型产气荚膜梭菌ETX编码基因进行优化设计,并将第106位组氨酸突变为脯氨酸,经人工合成获得基因片段GETXH106P。将GETXH106P克隆至原核表达载体p ET30a(+),转染BL21(DE3)菌中进行表达与纯化,从而获得重组蛋白r ETXH106P。利用Western blot方法检测r ETXH106P与D型产气荚膜梭菌抗血清的反应性,并检测其对犬肾细胞系(MDCK细胞)以及小鼠的毒力。随后,将r ETXH106P与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,按照《中华人民共和国兽药典》(2015年版)规定的方法检测一免及二免后兔血清的中和抗体效价。结果表明,r ETXH106P为可溶性表达,通过灰度扫描,其可溶性表达量比例可达30%;该重组蛋白能与D型产气荚膜梭菌毒素抗血清反应;细胞毒性试验结果显示,100μg/m L的重组蛋白仍无细胞毒性;小鼠安全性实验结果显示,6.25×106ng/kg剂量的重组蛋白对小鼠仍无致死性;每毫升的一免抗血清可中和450~700个最小致死量(MLD)、二免抗血清可中和3000~4000个MLD的D型产气荚膜梭菌毒素;用1 MLD的D型产气荚膜梭菌毒素攻毒后,对照组家兔4/4死亡,免疫组家兔4/4保护。以上结果表明,产气荚膜梭菌r ETXH106P重组蛋白毒力基本消失,且保留了良好的免疫原性,是D型产气荚膜梭菌病基因工程亚单位疫苗的理想候选抗原蛋白。

This experiment was conducted to obtain recombinant mutant of Clostridium perfringens ε toxin （ETX） and subsequently evaluate the virulence and immunogenicity of the recombinant toxin. The ETX gene of Clostridium perfringens type D strain, GETX H106P , using optimized codons was synthesized based on the sequence reported. At the same time, H106 was substituted with proline. Then, GETX H106P was cloned into prokaryotic expression vector pET30a-（＋） to get rETX H106P , following with purification. The reactivity of rETX H106P with antiserum of Clostridium perfringens type D was detected by Western blot. Meanwhile, Canine Kidney （MDCK） cell and mice were used to detect the virulence of rETX H106P . According to the method prescribed in Chinese Veterinary Pharmacopoeia （2015）, four rabbits were immunized with rETX H106P emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer of antiserums after first and twice immunization. The results showed that rETX H106P was expressed at a high level as soluble form with a ratio about 30% by gray scale scanning, and it could react with the antiserum of Clostridium perfringens type D. For MDCK cell, there were no apparent cytopathic effect （CPE） incubated with rETX H106P of 100μg/mL. At the same time, rETX H106P with the injection volume of 6.25×10 6 ng/kg was not fatal to ICR mice. After the first immunization, sera from rabbits immunized with rETX H106P could neutralize 450-700 Minimum Lethal Dose （MLD） Clostridium perfringens type D toxin per ml, and 3000-4000 mice MLD after twice immunization. Moreover, rabbits in rETX H106P immunized group fully survived at the dose of 1 MLD of Clostridium perfringens type D toxin challenge, whereas all of the rabbits died （4/4） in the control groups. The results suggest that rETX H106P without virulence retains the good immunogenic antigen, which is an ideal candidate antigen for genetic engineering subunit vaccine of Clostridium perfringens .