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

doi:10.3864/j.issn.0578-1752.2018.11.017

摘要/Abstract

摘要： 【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pET30a-(＋)后，将pET30a-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rETX_m2。利用Western blot方法，检测rETX_m2与D型产气荚膜梭菌ETX抗血清的反应性。将rETX_m2用细胞维持液稀释至100及10 μg·mL^-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rETX_m2，以0.0625、0.625和6.25 mg·kg^-1 3个剂量尾静脉注射，检测rETX_m2对小鼠的毒力。随后，将rETX_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），100 μg/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rETX_m2对家兔的免疫保护效果。【结果】在15℃和37℃条件，rETX_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rETX_m2。灰度扫描结果显示，在37℃诱导条件下，rETX_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rETX_m2，出现特异性反应条带。MDCK细胞毒性实验显示，在rETX_m2浓度为100 μg·mL^-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rETX_m2，对小鼠无致死性；血清中和试验结果显示，rETX_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rETX_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。

关键词: 产气荚膜梭菌&epsilon, 毒素, 突变, 重组表达, 毒力, 抗原性

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_m2activated 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_m2expressed 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_m2could 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

杜吉革，薛麒，朱真，李启红，印春生，姚文生，康凯，陈小云. 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[J]. 中国农业科学, 2018, 51(11): 2206-2215.

DU JiGe, XUE Qi, ZHU Zhen, LI QiHong, YIN ChunSheng, YAO WenSheng, KANG Kai, CHEN XiaoYun. Expression and Evaluation of Protective Efficacy of No-toxic Clostridium perfringens ε Toxin Derivative[J]. Scientia Agricultura Sinica, 2018, 51(11): 2206-2215.

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参考文献

[1] 郑晓丽, 窦贤明, 胡道俊, 肖勇, 赵翠荣, 倪学勤. 产气荚膜梭菌对养牛业的危害及其防制. 中国畜牧兽医, 2010, 37(8): 211-214.

ZHENG X L, DOU X M, HU D J, XIAO Y, ZHAO C R, NI X Q. The threat , prevention and control of Clostridium perfringens in cattle industry. China Animal Husbandry and Veterinary Medicine, 2010, 37(8):211-214. (in Chinese)

[2] 钏有科, 肖啸, 濮永华, 龚兴建. 努比亚山羊产气荚膜梭菌病的诊治. 中国兽医杂志, 2014, 50(12): 42-43.

CHUAN Y K, XIAO X, PU Y H, GONG X J. Diagnosis and treatment of Clostridium perfringens infection in Nubian goats. Chinese Journal of Veterinary Medicine, 2014, 50(12):42-43.(in Chinese)

[3] VAN I F, DE B J, PASMANS F, HUYGHEBAER G, HAESEBROUCK K, DUCATELLE R. Clostridium perfringens in poultry: an emerging threat for animal and public health. Avian Pathology, 2004, 33(6):537-549.

[4] 郑晓丽, 宋振银, 倪学勤. 产气荚膜梭菌对家禽业的危害及其预防.中国家禽, 2008, 30(24): 69-71.

ZHENG X L, SONG Z Y, NI X Q. Hazards and prevention of Clostridium perfringens to poultry industry. China Poultry, 2008, 30(24):69-71.(in Chinese)

[5] REVITT-MILLS S A, ROOD J I, ADAMS V. Clostridium perfringens extracellular toxins and enzymes: 20 and counting. Microbiology Australia, 2015, 36(3): 114-117.

[6] Li J H, Adams V, Bannam T L, Miyamoto K, Garcia J P, Uzal F A, Rood J L, McClanea B A. Toxin plasmids of clostridium perfringens. Microbiology and Molecular Biology Reviews, 2013, 77(2): 208-233.

[7] MCCLAIN M S, COVER T L. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin. Infection and Immunity, 2007, 75(4):1785-1793.

[8] LEWIS M, WEAVER C D, MCCLAIN M S. Identification of small molecule inhibitors of Clostridium perfringens epsilon-toxin cytotoxicity using a cell-based high-throughput screen. Toxins, 2010, 2(7):1825-1847.

[9] DORCA-AREVALO J, SOLER-JOVER A, GIBERT M, POPOFF M R, MARTIN-SATUE M, BLASI J. Binding of epsilon-toxin from Clostridium perfringens in the nervous system. Veterinary Microbiology, 2008, 131(1-2):14-25.

[10] POPOFF M R. Epsilon toxin: a fascinating pore-forming toxin. FEBS Journal, 2011, 278(23):4602-4615.

[11] FINNIE J W. Neurological disorders produced by Clostridium perfringens type D epsilon toxin. Anaerobe, 2004, 10(2): 145-150.

[12] SAYEED S, LI J H, MCCLANE B A. Virulence plasmid diversity in Clostridium perfringens type D isolates. Infection and Immunity, 2007, 75(5):2391-2398.

[13] HUNTER S E, CLARKE I N, KELLY D C, TITBALL R W. Cloning and nucleotide sequencing of the Clostridium perfringens epsilon- toxin gene and its expression in Escherichia coli. Infection and Immunity, 1992, 60(1):102-110.

[14] MINAMI J, KATAYAMA S, MATSUSHITA O, MATSUSHITA C, OKABE A. Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides. Microbiology and Immunology, 1997, 41(7):527-535.

[15] FREEDMAN J C, MCCLANE B A, UZAL F A. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia. Anaerobe, 2016, 41: 27-31.

[16] ALVES G G, RA M D Á, CHÁVEZOLÓRTEGUI C D, LOBATO F C F. Clostridium perfringens epsilon toxin: The third most potent bacterial toxin known. Anaerobe, 2014, 30:102-107.

[17] COLE A R, GIBERT M, POPOFF M, MOSS D S, TITBALL R W, BASAK A K. Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin. Nature Structural and Molecular Biology, 2004, 11(8):797-798.

[18] NAGAHAMA M, ODA M, KOBAYASHI K, OCHI S, TAKAGISHI T, SHIBUTANI M, SAKURAI J. A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens. Microbiology and Immunology, 2013, 57(5):340-345.

[19] DAS S, MAJUMDER S, KINGSTON J J, BATRA H V. Generation and characterization of recombinant bivalent fusion protein r-Cpib for immunotherapy against Clostridium perfringens beta and iota toxemia. Molecular Immunology, 2016, 70:140-148.

[20] ALIMOLAEI M, GOLCHIN M, DANESHVAR H. Oral immunization of mice against Clostridium perfringens, epsilon toxin with a Lactobacillus casei, vector vaccine expressing epsilon toxoid. Infection, Genetics and Evolution, 2016, 40:282-287.

[21] LI Q, XIN W, GAO S, KANG L, WANG J. A low-toxic site-directed mutant of Clostridium perfringens erfringens low-toxic site-directed mutant of Closenterotoxemia. Human Vaccines and Immunotherapeutics, 2013, 9(11):2386-2392.

[22] DORCA-ARÉVALO J, PAUILLAC S, DLAZ-HIDALGO L, MARTÍN-SATUÉ M, POPOFF M R, BLASI J. Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens. PLoS One, 2014, 9(7): e102417.

[23] IVIE S E, MCCLAIN M S. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1. Biochemistry, 2012, 51: 7588-7595.

[24] 李箐.产气荚膜梭菌α、ε毒素突变体构建及应用[D]. 安徽: 安徽医科大学, 2013.

LI Q. Construction of mutants of Clostridium perfringens Alpha, epsilon toxin and it’s application[D]. Anhui: Anhui medical university, 2013. (in Chinese)

[25] 罗维佳, 邓晨, 李衍常, 高媛, 徐平. 串联杂种泛素结合结构域蛋白(ThUBD)在大肠杆菌中的可溶性高效表达. 军事医学, 2016, 40(10):795-800.

LUO W J, DONG C, LI Y C, GAO Y, XU P. Soluble expression of tandem hybrid ubiquitin-binding domains（ThUBD）In prokaryotic cytoplasm of Escherichia coli BL21(DE3). Military Medical Sciences, 2016, 40(10):795-800. (in Chinese)

[26] 于蕊, 王双, 余云舟, 俞炜源, 孙志伟. B型肉毒毒素保护性抗原He在大肠杆菌中的可溶性高表达. 生物技术通讯, 2008, 19(3): 365-367.

YU R, WANG S, YU Y Z, YU W Y, SUN Z W. High-level and soluble expression of Botulinum Neurotoxin serotype B protective antigen HC domain in Escherichia coli. Letter in Biotechnology, 2008, 19(3):365-367. (in Chinese)

[27] 黄保英, 王文玲, 王秀平, 蒋涛, 谭文杰, 阮力. 甲型流感病毒核蛋白在大肠杆菌中的可溶性高效表达与纯化. 病毒学报, 2011(1):50-57.

HUANG B Y, WANG W L, WANG X P, JIANG T, TAN W J, RUAN L. Efficient soluble expression and purification of influenza A nucleoprotein in Escherichia coli. Chinese Journal of Virology, 2011(1): 50-57. (in Chinese)

[28] BOKORI-BROWN M, SAVVA C G, FERNANDES DA COSTA S P, NAYLOR C E, BASAK A K, TITBALL R W. Molecular basis of toxicity of Clostridium perfringens epsilon toxin. FEBS Journal, 2011, 278(23):4589-2011.

[29] 中国兽药典委员会. 中华人民共和国兽药典2015年版三部. 北京：中国农业出版社，2016:45-46.

Commission of Chinese Veterinary Pharmacopoeia. Veterinary Pharmacopoeia of the People’s Republic of China volume Ⅲ 2015 edition. Beijing: China Agricultural Press, 2016:45-46. (in Chinese)

[30] OYSTON P C F, PAYNE D W, HAVARD H L, WILLIANMSON D, TITBALL R W. Production of a non-toxic site-directed mutant of Clostridium perfringens epsilon-toxin which induces protective immunity in mice. Microbiology, 1998, 144 (2):333-341.

[31] 彭小兵, 田冬青, 彭国瑞, 李旭妮, 王蒙, 蒋玉文. 产气荚膜梭菌β毒素的表达及其抗血清的制备. 畜牧与兽医, 2015, 47(10): 93-96.

PENG X B, TIAN D Q, PENG G R, LI X N, WANG M, JIANG Y W. Expression of Clostridium perfringens β toxin and preparation of its antiserum. Animal Husbandry and Veterinary Medicine, 2015, 47(10): 93-96. (in Chinese)