Abstract: 【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 optimizedaccording 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 GETX_m. And this synthetic fragment was then cloned into prokaryotic expression vector pET30a-(＋) . Subsequently, pET30a- GETX_m2 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, rETX_m2. rETX_m2 expressed in a soluble form was then purified by Ni-IDA chromatograph. The reactivity of rETX_m2 with antiserum of Clostridium perfringens type D was detected by Western blot. Meanwhile, rETX_m2 was diluted with cell maintenance medium up to 100 and 10 μg·ml^-1 and then incubated with Canine Kidney (MDCK) cells to detect the cytotoxicity of it. Moreover, rETX_m2 and rETX_m2 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 rETX_m2 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 rETX_m2. 【Result】 rETX_m2 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, rETX_m2 expressed as soluble form induced at 37℃ was purified. The results of gray scale scanning showed that the rate of rETX_m2 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 rETX_m2 at a concentration of 100 μg·ml^-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, rETX_m2 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 rETX_m2 could neutralize 450-750 mice MLD Clostridium perfringens type D toxin per ml, and 2 500-4 000 mice MLD after twice immunization. Moreover, rabbits in rETX_m2 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 rETX_m2 without virulence retains the good immunogenic antigen, which is an ideal candidate antigen for genetic engineering subunit vaccine of Clostridium perfringens.

Key words: Clostridium perfringens &epsilon, toxin, mutation, recombinant expression, virulence, antigenicity