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
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摘要
为获得腐败梭菌α毒素和D型产气荚膜梭菌ε毒素的共表达产物,并鉴定其致死活性和反应原性,评价其免疫保护效力,利用PCR扩增腐败梭菌α毒素和产气荚膜梭菌ε毒素基因Gcsa和Gcpe并克隆至pETDuet-1质粒,转化E.coli BL21(DE3),自诱导培养基诱导这2段基因共表达;用SDS-PAGE和Western-blot以及小鼠毒性试验、抗毒素血清中和试验对表达产物进行鉴定;将诱导后的大肠杆菌灭活,制成铝佐剂疫苗1 m L/只皮下注射免疫家兔2次,测定免疫血清中和抗体效价,用1 MLD的腐败梭菌毒素和D型产气荚膜梭菌毒素分别攻击。结果显示,所构建的E.coli BL21(pETDuet-Gcsa-Gcpe)可以同时表达Gcsa和Gcpe,表达产物能够与腐败梭菌和D型产气荚膜抗毒素血清反应;且具有小鼠致死毒性,只有用2种抗毒素一同作用,才能将其毒性中和;制成灭活疫苗免疫家兔,免疫血清对腐败梭菌毒素的中和抗体效价为2 MLD/0.1 m L,对D型产气荚膜梭菌毒素的中和抗体效价≥50 MLD/0.1 m L;用2种毒素攻击,免疫组家兔全部保护,对照组全部死亡,达到《中华人民共和国兽药典》(2015版)效力检验标准。结果表明,所构建的表达载体可以实现α毒素和ε毒素的活性共表达,本研究为腐败梭菌和产气荚膜梭菌的多联基因工程灭活疫苗的研究提供了实验基础。
In order to prepare co-expression of Clostridium septium α-toxin and Clostridium perfringens( D) ε-toxin,identify its toxicity and reactionogenicity,and evaluate its immune efficacy,in this study,Gcsa encoding Clostridium septium α-toxin and Gcpe encoding Clostridium perfringens ε-toxin were amplified and cloned into the co-expression plasmid vector p ETDuet-1 respectively,and transformed into E.coli BL21( DE3). Gcsa and Gcpe were induced and co-expressed in medium for auto-induction,and the co-expression was identified with SDSPAGE and Western-blot,toxicity and neutralization test. Besides,the co-expression was inactivated and prepared into vaccine. Rabbits were immunized with 1 m L of the vaccine twice. And the immunological effect was evaluated by 1 MLD C. septium or C. perfringens( D) toxin challenge and serum neutralization tests. The results showed that E. coli BL21( p ETDuet-Gcsa-Gcpe) was constructed and used to co-expressed Gcsa and Gcpe successfully. And the co-expression was toxicant for mouse and could react with C.septium and C.perfringens( D)anti-toxoid sera peculiarly. Furthermore,only the two kinds of anti-toxin sera together could neutralize its toxicity. In addition,the co-expression vaccine generated 2 MLD and ≥50 MLD of neutralizing antibodies against respective toxin after the rabbits were immunized twice. And the rabbits were completely protected from the two kinds of toxin challenge,which reached to the principle of Chinese Veterinary Pharmacopoeia( 2015). The results suggested that C.septium α-toxin and C.perfringens ε-toxin can co-express in E.coli,and the co-expression may be developed into a bivalent vaccine candidate.
In order to prepare co-expression of Clostridium septium α-toxin and Clostridium perfringens( D) ε-toxin,identify its toxicity and reactionogenicity,and evaluate its immune efficacy,in this study,Gcsa encoding Clostridium septium α-toxin and Gcpe encoding Clostridium perfringens ε-toxin were amplified and cloned into the co-expression plasmid vector p ETDuet-1 respectively,and transformed into E.coli BL21( DE3). Gcsa and Gcpe were induced and co-expressed in medium for auto-induction,and the co-expression was identified with SDSPAGE and Western-blot,toxicity and neutralization test. Besides,the co-expression was inactivated and prepared into vaccine. Rabbits were immunized with 1 m L of the vaccine twice. And the immunological effect was evaluated by 1 MLD C. septium or C. perfringens( D) toxin challenge and serum neutralization tests. The results showed that E. coli BL21( p ETDuet-Gcsa-Gcpe) was constructed and used to co-expressed Gcsa and Gcpe successfully. And the co-expression was toxicant for mouse and could react with C.septium and C.perfringens( D)anti-toxoid sera peculiarly. Furthermore,only the two kinds of anti-toxin sera together could neutralize its toxicity. In addition,the co-expression vaccine generated 2 MLD and ≥50 MLD of neutralizing antibodies against respective toxin after the rabbits were immunized twice. And the rabbits were completely protected from the two kinds of toxin challenge,which reached to the principle of Chinese Veterinary Pharmacopoeia( 2015). The results suggested that C.septium α-toxin and C.perfringens ε-toxin can co-express in E.coli,and the co-expression may be developed into a bivalent vaccine candidate.
引文
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[6]Popoff M R.Epsilon toxin:a fascinating pore-forming toxin[J].FEBS Journal,2011,278(23):4602-4615.
[7]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研究[J].中国兽医杂志,2016,52(4):6-12.Peng X B,Li X N,Jiang Y W,et al.Study on relationship between antigenic amount of Clostridium Septicum toxoid and its immune effect[J].Chinese Journal of Veterinary Medicine.2016,52(04):6-12.
[8]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,2019,49(02):220-225.Peng G R,Peng X B,Li X N,et al.Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum[J].Chinese Veterinary Science,2019,49(02):220-225.
[9]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.
[10]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 J g,Zhang X k,Zhu Z,et al.Expression and immunocompetence of Clostridium perfringensεtoxin derivative with three mutations[J].Chinese journal of Veterinary Drug,2018,52(7):28-34.
[11]Alimolaei M,Golchin M,Daneshvar H.Oral immunization of mice against Clostridium perfringens,epsilon toxin with a Lactobacillus casei,vector vaccine expressing epsilon toxoid[J].Infection,Genetics and Evolution,2016,40:282-287.
[12]Dorca-arévalo J,Pauillac S,Dlaz-hidalgo L,et al.Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens[J].PLoSOne,2014,9(7):e102417.
[13]中国兽药典委员会.中华人民共和国兽药典2015年版三部[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 volumeⅢ2015edition[M].Beijing:China Agricultural Press,2016:45-46.
[14]Zhang Y,Qiao X,Yu X,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.
[15]Liu X L,Lin J,Hu H F,et al.Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli[J].Chinese Journal of Natural Medicines,2016,14(4):286-293.
[16]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.
[17]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):5978-85.
[18]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 G W,Li Z C,Gong X W,et al.Co-expression of Clostridium perfringensα-toxin andε-toxin and study on its immunogenicity[J].Chinese Veterinary Science,2014,44(6):630-635.
[19]Ferreira M R,Moreira G M,Cunha C E,et al.Recombinant alpha,beta,and epsilon toxins of Clostridium perfringens:production strategies and applications as veterinary vaccines[J].Toxins,2016,8(11).pii:E340.
[2]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 C8te d'Ivoire[J].Journal of Medical Primatology,2013,26:12047.
[3]彭国瑞,蒋玉文,彭小兵,等.腐败梭菌α毒素的研究进展[J].中国兽药杂志,2013,47(10):53-56.Peng G R,Jiang Y W,Peng X B,et al.Research Progress of Clostridium septicum Alpha-toxin[J].Chinese journal of Veterinary Drug,2013,47(10):53-56.
[4]Van I F,De B J,Pasmans F,et al.Clostridium perfringens in poultry:an emerging threat for animal and public health[J].Avian Pathology,2004,33(6):537-549.
[5]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.
[6]Popoff M R.Epsilon toxin:a fascinating pore-forming toxin[J].FEBS Journal,2011,278(23):4602-4615.
[7]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研究[J].中国兽医杂志,2016,52(4):6-12.Peng X B,Li X N,Jiang Y W,et al.Study on relationship between antigenic amount of Clostridium Septicum toxoid and its immune effect[J].Chinese Journal of Veterinary Medicine.2016,52(04):6-12.
[8]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,2019,49(02):220-225.Peng G R,Peng X B,Li X N,et al.Expression and immunogenicity evaluation of two truncated alpha toxins of Clostridium septicum[J].Chinese Veterinary Science,2019,49(02):220-225.
[9]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.
[10]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 J g,Zhang X k,Zhu Z,et al.Expression and immunocompetence of Clostridium perfringensεtoxin derivative with three mutations[J].Chinese journal of Veterinary Drug,2018,52(7):28-34.
[11]Alimolaei M,Golchin M,Daneshvar H.Oral immunization of mice against Clostridium perfringens,epsilon toxin with a Lactobacillus casei,vector vaccine expressing epsilon toxoid[J].Infection,Genetics and Evolution,2016,40:282-287.
[12]Dorca-arévalo J,Pauillac S,Dlaz-hidalgo L,et al.Correlation between in vitro cytotoxicity and in vivo lethal activity in mice of epsilon toxin mutants from Clostridium perfringens[J].PLoSOne,2014,9(7):e102417.
[13]中国兽药典委员会.中华人民共和国兽药典2015年版三部[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 volumeⅢ2015edition[M].Beijing:China Agricultural Press,2016:45-46.
[14]Zhang Y,Qiao X,Yu X,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.
[15]Liu X L,Lin J,Hu H F,et al.Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli[J].Chinese Journal of Natural Medicines,2016,14(4):286-293.
[16]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.
[17]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):5978-85.
[18]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 G W,Li Z C,Gong X W,et al.Co-expression of Clostridium perfringensα-toxin andε-toxin and study on its immunogenicity[J].Chinese Veterinary Science,2014,44(6):630-635.
[19]Ferreira M R,Moreira G M,Cunha C E,et al.Recombinant alpha,beta,and epsilon toxins of Clostridium perfringens:production strategies and applications as veterinary vaccines[J].Toxins,2016,8(11).pii:E340.
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