重组产气荚膜梭菌ε毒素三点突变体的融合表达及其免疫活性分析

Expression and Immunocompetence of Clostridium perfringens ε Toxin Derivative with Three mutations

关键词:  产气荚膜梭菌ε毒素  突变体  Th细胞表位  鞭毛蛋白  抗原性

Key words:  Clostridium perfringens ε toxin  mutation  Th cell epitope  flagellin  antigenicity

摘要:

为获得重组产气荚膜梭菌ε毒素（ETX）三点突变体蛋白，并评价其毒力及免疫活性。对已知的D型产气荚膜梭菌ETX编码基因进行优化设计，同时引入包括第30和196位酪氨酸突变为丙氨酸，及第106位组氨酸突变为脯氨酸共3个氨基酸突变。此外，在该基因5’端添加Th细胞表位（T）和鞭毛蛋白（flagellin）N末端编码序列，经人工合成获得基因片段GTFNETXm3。将该基因片段克隆至原核表达载体pET30a-(＋)，转染BL21（DE3）菌诱导表达、纯化，从而获得重组蛋白。利用Western blot方法检测重组蛋白与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）及小鼠的毒力。随后，将重组蛋白与Montanide ISA 201佐剂混合乳化制备疫苗，免疫4只健康家兔，根据《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。结果显示，重组蛋白主要以包涵体的形式存在，且能与D型产气荚膜梭菌毒素抗血清反应；100 μg/ml的重组蛋白未显示细胞毒性；6.25 mg/kg剂量的重组蛋白对小鼠无致死性；每毫升的一免抗血清可中和80~120个最小致死量（MLD）、二免抗血清可中和750～1100个MLD的D型产气荚膜梭菌毒素；用1个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。试验表明，产气荚膜梭菌ETX三点突变体的毒力基本消失，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。

Abstract:

This experiment was conducted to obtain Clostridium perfringens ETX derivative with three amino acid mutations, and subsequently the virulence and immunogenicity of the recombinant protein. Based on the known sequence, three amino acid mutations, Y30A, H106P and Y196A, were introduced into the gene of Clostridium perfringens ETX. Meanwhile, genes of Th cell and were added to 5’ of gene of CPA, respectively. Then the gene GTFNETXm3 was optimized and synthesized and subsequently cloned into prokaryotic expression vector pET-30a (＋) for expression and purification to get the recombinant protein. The reactivity of recombinant protein with antiserum of Clostridium perfringens type D was detected by Western blot. Meanwhile, the recombinant protein was incubated with Canine Kidney (MDCK) cells and injected into mice to detect the virulence of it. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), four rabbits were immunized with recombinant protein emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer of antiserums after the first and twice immunization. The results showed that the recombinant protein was presented predominantly in an insoluble form (inclusion bodies), and it could react with the antiserum of Clostridium perfringens type D. For MDCK cell, there were no apparent cytopathic effect (CPE) incubated with the recombinant protein of 100μg/ml. At the same time, the recombinant protein with the injection volume of 6.25 mg/kg was not fatal to ICR mice. After the first immunization, sera from rabbits immunized with the recombinant protein could neutralize 80-120 Minimum Lethal Dose (MLD) of Clostridium perfringens type D toxin per ml, and 750-1100 MLD after twice immunization. Moreover, rabbits in the recombinant protein 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 Clostridium perfringens ETX derivative with three mutations without virulence retains the good immunogenic antigen, which provides important experimental data for the development of novel Clostridium perfringens vaccine.