无毒性产气荚膜梭菌ε毒素突变体的表达及免疫保护力评价

Expression and Evaluation of Protective Efficacy of No-toxic Clostridium perfringens ε Toxin Derivative

【目的】获得产气荚膜梭菌ε毒素(ETX)的无毒重组突变体,并评价其毒力及免疫原性。【方法】对已知的D型产气荚膜梭菌ETX编码基因按照大肠杆菌偏爱密码子进行优化设计。同时,将第106位的组氨酸和第199位的苯丙氨酸分别突变为脯氨酸和谷氨酸,经人工合成,获得基因片段GETX_(m2)。将GETX_(m2)克隆至原核表达载体p ET30a-(+)后,将p ET30a-GETX_(m2)转化至BL21(DE3)感受态细胞中,在15℃和37℃两种条件下分别用IPTG诱导16 h和4 h,超声破碎后收集上清和沉淀,采用SDS-PAGE和Western blot方法检测重组蛋白的表达情况及其可溶性。利用Ni-IDA亲和层析方法对可溶性表达的重组蛋白进行纯化,从而获得重组蛋白r ETX_(m2)。利用Western blot方法,检测r ETX_(m2)与D型产气荚膜梭菌ETX抗血清的反应性。将r ETX_(m2)用细胞维持液稀释至100及10μg·m L^(-1),检测其对犬肾细胞系(MDCK细胞)的毒力。分别用胰酶活化前和活化后的r ETX_(m2),以0.0625、0.625和6.25 mg·kg^(-1) 3个剂量尾静脉注射,检测r ETX_(m2)对小鼠的毒力。随后,将r ETX_(m2)与Montanide ISA 201佐剂混合乳化制备疫苗,取4只健康家兔,皮下免疫2次(间隔两周),100μg/只。同时,Montanide ISA 201佐剂与PBS混合液免疫组作为对照组。分别在一免后14 d以及二免后21 d,采血,分离血清,按照《中华人民共和国兽药典》(2015年版)规定的方法检测血清对D型产气荚膜梭菌毒素的中和抗体效价。同时,在二免后21 d,对家兔经耳缘静脉注射1个家兔MLD的D型产气荚膜梭菌毒素,检测r ETX_(m2)对家兔的免疫保护效果。【结果】在15℃和37℃条件,r ETX_(m2)在BL21(DE3)菌体中均以可溶性和包涵体两种形式表达,综合考虑重组蛋白的表达量、可溶性及诱导时间,选择纯化在37℃条件下诱导获得的可溶性r ETX_(m2)。灰度扫描结果显示,在37℃诱导条件下,r ETX_(m2)可溶表达比例达30%;Western blot检测结果表明,D型产气荚膜梭菌毒素抗血清能够特异性的识别r ETX_(m2),出现特异性反应条带。MDCK细胞毒性实验显示,在r ETX_(m2)浓度为100μg·m L^(-1)的细胞培养基中孵育24 h后,细胞未出现细胞病变,而2 000倍稀释的D型产气荚膜梭菌天然毒素细胞培养基孵育组的细胞出现了明显的细胞病变;小鼠安全试验显示,尾静脉注射6.25 mg·kg^(-1)剂量的r ETX_(m2),对小鼠无致死性;血清中和试验结果显示,r ETX_(m2)免疫组每毫升的一免兔抗血清可中和450—750个小鼠MLD,每毫升的二免兔抗血清可中和2 500—4 000个小鼠MLD的D型产气荚膜梭菌毒素,而对照组的兔抗血清对D型产气荚膜梭菌毒素无中和作用;用1个家兔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。【结论】产气荚膜梭菌r ETX_(m2)毒力基本丧失,但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。

【Objective】This experiment was conducted to obtain no-toxic Clostridium perfringens ε toxin（ETX） derivative and subsequently evaluate the virulence and immunogenicity of it. 【Method】The ETX gene of Clostridium perfringens type D strain was optimized according to Escherichia coli（E. coli） expression system codon preferences. At the same time, H106 and F199 were substituted with proline and glutamic acid, respectively, following with synthesis of GETXm. And this synthetic fragment was then cloned into prokaryotic expression vector p ET30 a-（＋）. Subsequently, p ET30 a-GETXm2 was transformed into BL21（DE3） competent cells and induced by IPTG at 15℃ and 37℃ for 16 h and 4 h, respectively. The supernatant and the precipitate of the cells broken by ultrasonic were collected and subjected to SDS-PAGE and Western blot to detect the expression and solubility of recombinant protein, r ETXm2. r ETXm2 expressed in a soluble form was then purified by Ni-IDA chromatograph. The reactivity of r ETXm2 with antiserum of Clostridium perfringens type D was detected by Western blot. Meanwhile, r ETXm2 was diluted with cell maintenance medium up to 100 and 10 μg·m L-1 and then incubated with Canine Kidney（MDCK） cells to detect the cytotoxicity of it. Moreover, r ETXm2 and r ETXm2 activated by trypsin were tested for the virulence to mice by tail vein injection at doses of 0.0625, 0.625 and 6.25 mg·kg-1, respectively. According to the method prescribed in Chinese Veterinary Pharmacopoeia（2015）, four rabbits were immunized subcutaneously with 100 μg of r ETXm2 emulsified with oil adjuvant of ISA 201 twice（at an interval of 2 weeks）. Meanwhile, rabbits of adjuvant control group were immunized with mixture of Montanide ISA 201 adjuvant and PBS. Serum samples were collected 14 d after the first immunization and 21 d after last immunization to detect the neutralizing titer against the Clostridium perfringens type D toxin. At the same time, rabbits were challenged with 1 rabbit MLD Clostridium perfringens type D toxin through the ear marginal veins 21 days after the second immunization to detect the protective efficacy of r ETXm2. 【Result】 r ETXm2 was expressed in both soluble and insoluble form（inclusion bodies） after induced at 15℃ and 37℃. Considering the expression level, solubility and time of induction, r ETXm2 expressed as soluble form induced at 37℃ was purified. The results of gray scale scanning showed that the rate of r ETXm2 expressed as soluble form was up to 30%. And it could react with the antiserum of Clostridium perfringens type D with specific band. Cytotoxicity assays showed that there was no cytopathic effect（CPE） after incubation in cell culture medium with r ETXm2 at a concentration of 100 μg·m L-1 for 24 h, whereas Clostridium perfringens Type D toxin with 2 000-fold dilution induced apparent CPE, characterized by predominant lysis. At the same time, r ETXm2 with the injection volume of 6.25 mg·kg-1 still was not fatal to mice. After the first immunization, sera from rabbits immunized with r ETXm2 could neutralize 450-750 mice MLD Clostridium perfringens type D toxin per m L, and 2 500-4 000 mice MLD after twice immunization. Moreover, rabbits in r ETXm2 immunized group fully survived at the dose of 1 rabbit MLD of Clostridium perfringens type D toxin challenge, whereas all of the rabbits died（4/4） in the control groups. 【Conclusion】The results suggest that r ETXm2 without virulence retains the good immunogenic antigen, which is an ideal candidate antigen for genetic engineering subunit vaccine of Clostridium perfringens.