重组腐败梭菌α毒素的制备与免疫效力评价
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摘要
为了对重组腐败梭菌α毒素的免疫效力进行评价,本研究以表达重组腐败梭菌α毒素的大肠杆菌BL21(pET28a-CSA)为研究对象,对影响重组腐败梭菌α毒素表达量的条件优化后,将重组腐败梭菌α毒素(分为上清分泌形式与包涵体形式)、超声波裂解和未裂解诱导后的全菌制成铝佐剂灭活疫苗皮下注射免疫家兔后,检测家兔血清中和效价,利用1 MLD腐败梭菌毒素攻击免疫家兔。结果显示,pET28a-CSA/BL21培养至OD_(600nm)达到0.850～0.950时以0.40 mmol/L IPTG,37℃诱导6 h获得的重组腐败梭菌α毒素相对表达量较高,并且以包涵体和可溶性2种形式表达且以包涵体居多;除包涵体疫苗组血清中和抗体效价为6 MLD/0.1 mL外,其它可溶性、裂解菌苗和未裂解处理菌苗实验组均为8 MLD/0.1 mL,高于类毒素疫苗组和《中国兽药典》规定的1 MLD/0.1 mL,攻毒后各免疫组家兔均得到100%保护,而类毒素疫苗组保护率仅40%,空白对照组全部死亡。结果表明,本研究制备的重组腐败梭菌α毒素具有很好的免疫保护效果,尤以可溶性的重组腐败梭菌α毒素免疫保护最佳。本实验为羊快疫疫苗的研制提供了实验基础。
To evaluate immune efficacy of recombinant alpha toxin of Clostridium septicum(rCSA) in rabbits, the expression conditions of the recombinant alpha toxin from recombinant Escherichia coli BL21(pET28 a-CSA) were optimized, and the soluble rCSA, inclusion body rCSA, lysed bacterium and untreated bacterium were collected and inactivated with 0.2% of formaldehyde,respectively. Then rabbits were immunized by these rCSA in different forms and challenged by the alpha toxin of C.septicum, and serum neutralization antibody titers were tested in mice. The results showed that the rCSA production was higher, when the E.coli BL21(pET28a-CSA) was cultured for 4 hrs at the OD_(600nm) ranged from 0.850 to 0.950 and then induced for 6 hrs with 0.40 mmol/L of IPTG at 37℃. The serum neutralization titers of in the rabbits in immunized with the soluble, lysed bacterium and untreated bacterium groups were all 8 MLD/0.1 mL, and inclusions group was 6 MLD/0.1 mL. However, their titers were higher than 1 MLD/0.1 mL of toxoid vaccine group and the titer of qualified vaccine prescribed by Chinese Veterinary Pharmacopoeia. And 5/5 rabbits immunized with rCSA survived at the dose of 1 MLD toxin of C.septicum challenge, whereas only 2/5 rabbits immunized by toxoid vaccine survived, and all of the rabbits in control group died. The results suggested that the rCSA of C.septicum had strong immunogenicity, and this study provides experimental data for preparation and evaluation immune efficacy in sheep of the promising candidate Braxy vaccine.
To evaluate immune efficacy of recombinant alpha toxin of Clostridium septicum(rCSA) in rabbits, the expression conditions of the recombinant alpha toxin from recombinant Escherichia coli BL21(pET28 a-CSA) were optimized, and the soluble rCSA, inclusion body rCSA, lysed bacterium and untreated bacterium were collected and inactivated with 0.2% of formaldehyde,respectively. Then rabbits were immunized by these rCSA in different forms and challenged by the alpha toxin of C.septicum, and serum neutralization antibody titers were tested in mice. The results showed that the rCSA production was higher, when the E.coli BL21(pET28a-CSA) was cultured for 4 hrs at the OD_(600nm) ranged from 0.850 to 0.950 and then induced for 6 hrs with 0.40 mmol/L of IPTG at 37℃. The serum neutralization titers of in the rabbits in immunized with the soluble, lysed bacterium and untreated bacterium groups were all 8 MLD/0.1 mL, and inclusions group was 6 MLD/0.1 mL. However, their titers were higher than 1 MLD/0.1 mL of toxoid vaccine group and the titer of qualified vaccine prescribed by Chinese Veterinary Pharmacopoeia. And 5/5 rabbits immunized with rCSA survived at the dose of 1 MLD toxin of C.septicum challenge, whereas only 2/5 rabbits immunized by toxoid vaccine survived, and all of the rabbits in control group died. The results suggested that the rCSA of C.septicum had strong immunogenicity, and this study provides experimental data for preparation and evaluation immune efficacy in sheep of the promising candidate Braxy vaccine.
引文
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[9]张志强,杨楠,李永慧,等.迟缓爱德华菌外膜蛋白OmpA原核表达及其免疫原性研究[J].中国预防兽医学报,2018,40(6):534-537
[10]刘雪慧,王方山,樊金,等.D型产气荚膜梭菌类毒素疫苗的研制及保护效果的评价[J].中国预防兽医学报,2015,37(4):310-314.
[11]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]Oliver K,Elke M,Sanae B M,et al.Clostridium septicum alpha-toxin forms pores and induces rapid cell necrosis[J].Toxicon,2010,55(1):61-72.
[3]Mukamoto M,Kimura R,Hang'ombe M B,et al.Analysis of tryptophan-rich region in Clostridium septicum alpha-toxin involved with binding to glycosylphosphatidylinositol-anchored proteins[J].Microbiol Immunol,2013,57(3):163-169.
[4]Chakravorty A,Awad M M,Cheung J K,et al.The pore-formingα-toxin from Clostridium septicum activates the MAPK pathway in a Ras-c-Raf-dependent and independent manner[J].Toxins(Basel),2015,7(2):516-534.
[5]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):6-8,12.
[6]Amimoto K,Sasaki Y,Fukuyama S,et al.Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin[J].Vet Microbiol,2006,114(1-2):51-59.
[7]Lancto C A,Foster L K,Kromm M M,et al.A noncytolyticαtoxin recombinant protein protects turkeys against Clostridium septicum challenge[J].Avian Dis,2014,58(4):566-571.
[8]彭国瑞.重组腐败梭菌α毒素的免疫原性研究[D].北京:中国兽医药品监察所,2014.
[9]张志强,杨楠,李永慧,等.迟缓爱德华菌外膜蛋白OmpA原核表达及其免疫原性研究[J].中国预防兽医学报,2018,40(6):534-537
[10]刘雪慧,王方山,樊金,等.D型产气荚膜梭菌类毒素疫苗的研制及保护效果的评价[J].中国预防兽医学报,2015,37(4):310-314.
[11]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.