产气荚膜梭菌β毒素和ε毒素以及腐败梭菌α毒素三联基因工程灭活疫苗的制备与免疫效力评价
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摘要
为了获得高表达量的重组C型产气荚膜梭菌β毒素,鉴定其致死性和反应原性,并评价其与D型产气荚膜梭菌ε毒素和腐败梭菌α毒素共表达产物联合制成三联基因工程灭活疫苗的免疫保护效力。设计含不同酶切位点的2对引物,PCR扩增C型产气荚膜梭菌β毒素基因cpb1和cpb2,将二者克隆至pRSFDuet-1质粒,转化大肠杆菌BL21 (DE3),自诱导培养基诱导2段基因共表达。用SDS-PAGE和Western-blot以及小鼠毒性试验、抗毒素血清和中和试验对表达产物进行鉴定。将诱导收获的大肠杆菌以及D型产气荚膜梭菌ε毒素和腐败梭菌α毒素的共表达产物1∶2配比灭活制成三联灭活铝佐剂疫苗,以每只1 m L剂量皮下免疫家兔2次后,测定血清中和抗体效价,用1 MLD的C、D型产气荚膜梭菌毒素和腐败梭菌毒素分别攻击。结果显示,所构建的重组大肠杆菌BL21(pRSFDuet-cpb1-cpb2)能够表达重组β毒素,产物能够与C型产气荚膜梭菌抗毒素血清反应,且具有小鼠致死毒性,抗毒素血清可以将其毒性中和;制成联合疫苗免疫家兔,免疫血清对C型产气荚膜梭菌毒素的中和抗体效价为8 MLD/0.1 m L,对D型产气荚膜梭菌毒素的中和抗体效价为45 MLD/0.1 m L,对腐败梭菌毒素的中和抗体效价为1.5 MLD/0.1 m L;用3种毒素攻击免疫组家兔均全部保护,对照组全部死亡,达到《中华人民共和国兽药典三部(2015版)》效力检验标准。本研究为羊猝狙、肠毒血症和快疫三联疫苗的研制提供了试验基础。
In order to acquire high level co-expression of Clostridium perfringens(C) β-toxin,identify its toxicity and reactionogenicity,and evaluate immune efficacy of genetic engineering triple vaccine of co-expressions of C.perfringens(C) β-toxin,C.perfringens(D) ε-toxin and C.septicum α-toxin,two pairs of primers were designed to amplify cpb1 and cpb2 genes,which contained the same C.perfringens(C) β-toxin gene and different restriction sites.The two genes were cloned into the plasmid vector p RSFDuet-1,and the recombinant plasmids were transferred into Escherichia coli BL21.Cpb1 and cpb2 were induced and co-expressed in medium for auto-induction.The toxicity and reactionogenicity of the co-expression were identified.After induction and inactivation,this induced products and co-expression of C.perfringens(D) ε-toxin and C.septicum α-toxin prepared before were mixed with an adjuvant aluminum hydroxide solution and prepared into triple vaccine.Rabbits were immunized twice with the vaccine.The immune effect was evaluated by C.perfringens(C),(D) or C.septium toxin challenge and serum neutralization tests.The results showed that recombinant β-toxin was expressed effectively in E.coli BL21(p RSFDuet-cpb1-cpb2),and could react with the antiserum of C.perfringens(C),and kill the mice.Its toxicity could be neutralized by C.perfringens(C) anti-toxin sera.The immune serum neutralization titers of the rabbits in the immune group were 8 MLD/0.1 m L for C.perfringens(C)toxin,45 MLD/0.1 m L for C.perfringens(D) toxin and 1.5 MLD/0.1 m L for C.septium toxin.The rabbits in the immune group all survived,when challenged with 1 MLD C.perfringens(C),(D) or C.septium toxin,whereas the rabbits in the negative control groups all died.The above-mentioned results were in accordance with the standard requirement prescribed in the Chinese Veterinary Pharmacopoeia(2015),and suggested that the co-expression may be developed into a triple vaccine candidate.
In order to acquire high level co-expression of Clostridium perfringens(C) β-toxin,identify its toxicity and reactionogenicity,and evaluate immune efficacy of genetic engineering triple vaccine of co-expressions of C.perfringens(C) β-toxin,C.perfringens(D) ε-toxin and C.septicum α-toxin,two pairs of primers were designed to amplify cpb1 and cpb2 genes,which contained the same C.perfringens(C) β-toxin gene and different restriction sites.The two genes were cloned into the plasmid vector p RSFDuet-1,and the recombinant plasmids were transferred into Escherichia coli BL21.Cpb1 and cpb2 were induced and co-expressed in medium for auto-induction.The toxicity and reactionogenicity of the co-expression were identified.After induction and inactivation,this induced products and co-expression of C.perfringens(D) ε-toxin and C.septicum α-toxin prepared before were mixed with an adjuvant aluminum hydroxide solution and prepared into triple vaccine.Rabbits were immunized twice with the vaccine.The immune effect was evaluated by C.perfringens(C),(D) or C.septium toxin challenge and serum neutralization tests.The results showed that recombinant β-toxin was expressed effectively in E.coli BL21(p RSFDuet-cpb1-cpb2),and could react with the antiserum of C.perfringens(C),and kill the mice.Its toxicity could be neutralized by C.perfringens(C) anti-toxin sera.The immune serum neutralization titers of the rabbits in the immune group were 8 MLD/0.1 m L for C.perfringens(C)toxin,45 MLD/0.1 m L for C.perfringens(D) toxin and 1.5 MLD/0.1 m L for C.septium toxin.The rabbits in the immune group all survived,when challenged with 1 MLD C.perfringens(C),(D) or C.septium toxin,whereas the rabbits in the negative control groups all died.The above-mentioned results were in accordance with the standard requirement prescribed in the Chinese Veterinary Pharmacopoeia(2015),and suggested that the co-expression may be developed into a triple vaccine candidate.
引文
[1]VAN IMMERSEEL F,DE JEROEN B,PASMANS F,et al.Clostridium perfringens in poultry:an emerging threat for animal and public health[J].Avian Pathology,2004,33(6):537-549.
[2]GARCIA J P,BEINGESSER J,FISHER D J,et al.The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats[J].Veterinary Microbiology,2012,157(3/4):412-419.
[3]FILHO E J,CARVALHO A U,ASSIS R A,et al.Clinicopathological features of experimental Clostridium perfringens type D enterotoxemia in cattle[J].Veterinary Pathology,2009,469(6):1213-1220.
[4]POPOFF M R.Epsilon toxin:A fascinating pore-forming toxin[J].FEBS Journal,2011,278(23):4602-4615.
[5]KENNEDY C L,LYRAS D,CORDNER L M,et al.Pore-forming activity of alpha-toxin is essential for Clostridium septicum-mediated myonecrosis[J].Infection and Immunity,2009,77(3):943-951.
[6]KIRCHNER S,M?TZ RENSING K,DORNER M B,et al.Necrotizing endometritis and isolation of an alpha-toxin producing strain of Clostridium septicum in a wild sooty mangabey from C?te d'Ivoire[J].Journal of Medical Primatology,2013,42(4):220-224.
[7]彭国瑞,彭小兵,李旭妮,等.截短腐败梭菌α毒素的表达及其免疫原性评价[J].中国兽医科学,2019,49(2):220-225.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum[J].Chinese Veterinary Science,2019,49(2):220-225.(in Chinese)
[8]彭小兵,李旭妮,蒋玉文,等.腐败梭菌类毒素抗原含量与免疫效果之间的关系试验[J].中国兽医杂志,2016,52(4):6-8,12.PENG Xiao-bing,LI Xu-ni,JIANG Yu-wen,et al.Study on relationship between antigenic amount of Clostridium septicum toxoid and its immune effect[J].Chinese Journal of Veterinary Medicine,2016,52(4):6-8,12.(in Chinese)
[9]彭小兵,田冬青,彭国瑞,等.产气荚膜梭菌β毒素的表达及其抗血清的制备[J].畜牧与兽医,2015,47(10):93-96.PENG Xiao-bing,TIAN Dong-qing,PENG Guo-rui,et al.Expression and serum preparation of Clostridium perfringensβtoxin[J].Animal Husbandry and Veterinary Medicine,2015,47(10):93-96.(in Chinese)
[10]SALVARAN F M,CONCEIO F R,CUNHA C E,et al.Vaccination with recombinant Clostridium perfringens toxoidsαandβpromotes elevated antepartum and passive humoral immunity in swine[J].Vaccine,2013,31(38):4152-4155.
[11]杜吉革,张秀坤,朱真,等.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[J].中国兽药杂志,2018,52(7):28-34.DU Ji-ge,ZHANG Xiu-kun,ZHU Zhen,et al.Expression and immunocompetence of Clostridium perfringensεtoxin derivative with three mutations[J].Chinese Journal of Veterinary Drug,2018,52(7):28-34.(in Chinese)
[12]DORCAARéVALO J,PAUILLAC S,D魱AZHIDALGO L,et al.Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens[J].PLoS One,2014,9(7):e102417.
[13]彭国瑞,彭小兵,李旭妮,等.重组腐败梭菌α毒素的生物活性及其灭活疫苗的研究[J].中国兽医学报,2019,39(6):1118-1124.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Biological activity of recombinant alpha toxin of Clostridium septicum and its inactivated vaccine[J].Chinese Journal of Veterinary Science,2019,39(6):1118-1124.(in Chinese)
[14]LANCTO C A,FOSTER L K,KROMM M M,et al.A noncytolyticαtoxin recombinant protein protects turkeys against Clostridium septicum challenge[J].Avian Diseases,2014,58(4):566-571.
[15]彭国瑞,彭小兵,李旭妮,等.腐败梭菌α毒素和产气荚膜梭菌ε毒素的共表达及其免疫效力评价[J].中国兽药杂志,2019,53(2):26-32.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Evaluation immune efficacy of co-expression of Clostridium septiumα-toxin and Clostridium perfringensε-toxin[J].Chinese Journal of Veterinary Drug,2019,53(2):26-32.(in Chinese)
[16]中国兽药典委员会.中华人民共和国兽药典三部(2015年版)[M].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 VolumeⅢ(2015 edition)[M].Beijing:China Agricultural Press,2016:45-46.(in Chinese)
[17]ZHANG Y P,QIAO X W,YU X M,et al.Enhanced soluble production of cholera toxin B subunit in Escherichia coli by co-expression of SKP chaperones[J].Protein Expression and Purification,2017,138:1-6.
[18]LIU X L,LIN J,HU H F,et al.Enhanced production of shikimic acid using a multi-gene co-ex pression system in Escherichia coli[J].Chinese Journal of Natural Medicines,2016,14(4):286-293.
[19]LU X Y,HE S Y,ZONG H,et al.Improved 1,2,4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins[J].World Journal of Microbiology and Biotechnology,2016,32(9):149.
[20]YU Y Z,GONG Z W,MA Y,et al.Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate[J].Vaccine,2011,29(35):5978-5985.
[21]韩广伟,李兆才,宫晓炜,等.产气荚膜梭菌α毒素和ε毒素的共表达及其免疫原性的研究[J].中国兽医科学,2014,44(6):630-635.HAN Guang-wei,LI Zhao-cai,GONG Xiao-wei,et al.Co-expression of Clostridium perfringensα-toxin andε-toxin and study on its immunogenicity[J].Chinese Veterinary Science,2014,44(6):630-635.(in Chinese)
[2]GARCIA J P,BEINGESSER J,FISHER D J,et al.The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats[J].Veterinary Microbiology,2012,157(3/4):412-419.
[3]FILHO E J,CARVALHO A U,ASSIS R A,et al.Clinicopathological features of experimental Clostridium perfringens type D enterotoxemia in cattle[J].Veterinary Pathology,2009,469(6):1213-1220.
[4]POPOFF M R.Epsilon toxin:A fascinating pore-forming toxin[J].FEBS Journal,2011,278(23):4602-4615.
[5]KENNEDY C L,LYRAS D,CORDNER L M,et al.Pore-forming activity of alpha-toxin is essential for Clostridium septicum-mediated myonecrosis[J].Infection and Immunity,2009,77(3):943-951.
[6]KIRCHNER S,M?TZ RENSING K,DORNER M B,et al.Necrotizing endometritis and isolation of an alpha-toxin producing strain of Clostridium septicum in a wild sooty mangabey from C?te d'Ivoire[J].Journal of Medical Primatology,2013,42(4):220-224.
[7]彭国瑞,彭小兵,李旭妮,等.截短腐败梭菌α毒素的表达及其免疫原性评价[J].中国兽医科学,2019,49(2):220-225.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum[J].Chinese Veterinary Science,2019,49(2):220-225.(in Chinese)
[8]彭小兵,李旭妮,蒋玉文,等.腐败梭菌类毒素抗原含量与免疫效果之间的关系试验[J].中国兽医杂志,2016,52(4):6-8,12.PENG Xiao-bing,LI Xu-ni,JIANG Yu-wen,et al.Study on relationship between antigenic amount of Clostridium septicum toxoid and its immune effect[J].Chinese Journal of Veterinary Medicine,2016,52(4):6-8,12.(in Chinese)
[9]彭小兵,田冬青,彭国瑞,等.产气荚膜梭菌β毒素的表达及其抗血清的制备[J].畜牧与兽医,2015,47(10):93-96.PENG Xiao-bing,TIAN Dong-qing,PENG Guo-rui,et al.Expression and serum preparation of Clostridium perfringensβtoxin[J].Animal Husbandry and Veterinary Medicine,2015,47(10):93-96.(in Chinese)
[10]SALVARAN F M,CONCEIO F R,CUNHA C E,et al.Vaccination with recombinant Clostridium perfringens toxoidsαandβpromotes elevated antepartum and passive humoral immunity in swine[J].Vaccine,2013,31(38):4152-4155.
[11]杜吉革,张秀坤,朱真,等.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[J].中国兽药杂志,2018,52(7):28-34.DU Ji-ge,ZHANG Xiu-kun,ZHU Zhen,et al.Expression and immunocompetence of Clostridium perfringensεtoxin derivative with three mutations[J].Chinese Journal of Veterinary Drug,2018,52(7):28-34.(in Chinese)
[12]DORCAARéVALO J,PAUILLAC S,D魱AZHIDALGO L,et al.Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens[J].PLoS One,2014,9(7):e102417.
[13]彭国瑞,彭小兵,李旭妮,等.重组腐败梭菌α毒素的生物活性及其灭活疫苗的研究[J].中国兽医学报,2019,39(6):1118-1124.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Biological activity of recombinant alpha toxin of Clostridium septicum and its inactivated vaccine[J].Chinese Journal of Veterinary Science,2019,39(6):1118-1124.(in Chinese)
[14]LANCTO C A,FOSTER L K,KROMM M M,et al.A noncytolyticαtoxin recombinant protein protects turkeys against Clostridium septicum challenge[J].Avian Diseases,2014,58(4):566-571.
[15]彭国瑞,彭小兵,李旭妮,等.腐败梭菌α毒素和产气荚膜梭菌ε毒素的共表达及其免疫效力评价[J].中国兽药杂志,2019,53(2):26-32.PENG Guo-rui,PENG Xiao-bing,LI Xu-ni,et al.Evaluation immune efficacy of co-expression of Clostridium septiumα-toxin and Clostridium perfringensε-toxin[J].Chinese Journal of Veterinary Drug,2019,53(2):26-32.(in Chinese)
[16]中国兽药典委员会.中华人民共和国兽药典三部(2015年版)[M].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 VolumeⅢ(2015 edition)[M].Beijing:China Agricultural Press,2016:45-46.(in Chinese)
[17]ZHANG Y P,QIAO X W,YU X M,et al.Enhanced soluble production of cholera toxin B subunit in Escherichia coli by co-expression of SKP chaperones[J].Protein Expression and Purification,2017,138:1-6.
[18]LIU X L,LIN J,HU H F,et al.Enhanced production of shikimic acid using a multi-gene co-ex pression system in Escherichia coli[J].Chinese Journal of Natural Medicines,2016,14(4):286-293.
[19]LU X Y,HE S Y,ZONG H,et al.Improved 1,2,4-butanetriol production from an engineered Escherichia coli by co-expression of different chaperone proteins[J].World Journal of Microbiology and Biotechnology,2016,32(9):149.
[20]YU Y Z,GONG Z W,MA Y,et al.Co-expression of tetanus toxin fragment C in Escherichia coli with thioredoxin and its evaluation as an effective subunit vaccine candidate[J].Vaccine,2011,29(35):5978-5985.
[21]韩广伟,李兆才,宫晓炜,等.产气荚膜梭菌α毒素和ε毒素的共表达及其免疫原性的研究[J].中国兽医科学,2014,44(6):630-635.HAN Guang-wei,LI Zhao-cai,GONG Xiao-wei,et al.Co-expression of Clostridium perfringensα-toxin andε-toxin and study on its immunogenicity[J].Chinese Veterinary Science,2014,44(6):630-635.(in Chinese)
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