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Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies.
BMC Biotechnology ( IF 3.5 ) Pub Date : 2020-01-28 , DOI: 10.1186/s12896-019-0597-4
Katia Forti 1, 2 , Monica Cagiola 1 , Martina Pellegrini 1 , Lucia Anzalone 1 , Antonella Di Paolo 1 , Sara Corneli 1 , Giulio Severi 1 , Antonio De Giuseppe 1
Affiliation  

BACKGROUND Clostridium perfringens is the causative agent of several diseases and enteric infections in animals and humans. The virulence of C. perfringens is largely attributable to the production of numerous toxins; of these, the alpha toxin (CPA) plays a crucial role in histotoxic infections (gas gangrene). CPA toxin consists of two domains, i.e., the phospholipase C active site, which lies in the N-terminal domain amino acid (aa residues 1-250), and the C-terminal region (aa residues 251-370), which is responsible for the interaction of the toxin with membrane phospholipids in the presence of calcium ions. All currently produced clostridial vaccines contain toxoids derived from culture supernatants that are inactivated, mostly using formalin. The CPA is an immunogenic antigen; recently, it has been shown that mice that were immunized with the C-terminal domain of the toxin produced in E. coli were protected against C. perfringens infections and the anti-sera produced were able to inhibit the CPA activity. Monoclonal and polyclonal antibodies were produced only against full-length CPA and not against the truncated forms. RESULTS In the present study, we have reported for the first time; about the generation of a recombinant baculovirus capable of producing a deleted rCPA toxin (rBacCPA250-363H6) lacking the N-terminal domain and the 28 amino acids (aa) of the putative signal sequence. The insertion of the L21 consensus sequence upstream of the translational start codon ATG, drastically increases the yield of recombinant protein in the baculovirus-based expression system. The protein was purified by Ni-NTA affinity chromatography and the lack of toxicity in vitro was confirmed in CaCo-2 cells. Polyclonal antibodies and eight hybridoma-secreting Monoclonal antibodies were generated and tested to assess specificity and reactivity. The anti-sera obtained against the fragment rBacCPA250-363H6 neutralized the phospholipase C activity of full-length PLC. CONCLUSIONS The L21 leader sequence enhanced the expression of atoxic C-terminal recombinant CPA protein produced in insect cells. The monoclonal and polyclonal antibodies obtained were specific and highly reactive. The availability of these biologicals could contribute to the development of diagnostic assays and/or new recombinant protein vaccines.

中文翻译:

表达产气荚膜梭菌无毒C末端CPA毒素的重组杆状病毒的产生和特异性抗体的产生。

背景技术产气荚膜梭菌是动物和人类中几种疾病和肠感染的病原体。产气荚膜梭菌的毒力在很大程度上归因于多种毒素的产生。其中,α毒素(CPA)在组织毒性感染(坏疽性气体)中起着至关重要的作用。CPA毒素由两个域组成,即位于N末端域氨基酸(氨基酸残基1-250)和C末端区域(氨基酸残基251-370)的磷脂酶C活性位点。在钙离子存在下毒素与膜磷脂的相互作用。目前所有生产的梭菌疫苗均含有来源于培养上清液的类毒素,该类上清液大多经福尔马林灭活。CPA是一种免疫原性抗原;最近,已经表明,用在大肠杆菌中产生的毒素的C-末端结构域免疫的小鼠被保护免受产气荚膜梭菌感染,并且产生的抗血清能够抑制CPA活性。单克隆和多克隆抗体仅针对全长CPA产生,而不针对截短形式。结果在本研究中,我们是首次报道。关于重组杆状病毒的产生,该杆状病毒能够产生缺失的cCPA毒素(rBacCPA250-363H6),该毒素缺乏N端结构域和推定信号序列的28个氨基酸(aa)。L21共有序列在翻译起始密码子ATG上游的插入大大增加了基于杆状病毒的表达系统中重组蛋白的产量。该蛋白通过Ni-NTA亲和色谱纯化,并在CaCo-2细胞中证实了体外无毒性。产生多克隆抗体和八种分泌杂交瘤的单克隆抗体,并进行测试以评估特异性和反应性。针对片段rBacCPA250-363H6获得的抗血清中和了全长PLC的磷脂酶C活性。结论L21前导序列增强了昆虫细胞中产生的无毒C末端重组CPA蛋白的表达。获得的单克隆抗体和多克隆抗体是特异性的且具有高反应性。这些生物制剂的可用性可能有助于诊断分析和/或新的重组蛋白疫苗的开发。产生多克隆抗体和八种分泌杂交瘤的单克隆抗体,并进行测试以评估特异性和反应性。针对片段rBacCPA250-363H6获得的抗血清中和了全长PLC的磷脂酶C活性。结论L21前导序列增强了昆虫细胞中产生的无毒C末端重组CPA蛋白的表达。获得的单克隆抗体和多克隆抗体是特异性的且具有高反应性。这些生物制剂的可用性可能有助于诊断分析和/或新的重组蛋白疫苗的开发。产生多克隆抗体和八种分泌杂交瘤的单克隆抗体,并进行测试以评估特异性和反应性。针对片段rBacCPA250-363H6获得的抗血清中和了全长PLC的磷脂酶C活性。结论L21前导序列增强了昆虫细胞中产生的无毒C末端重组CPA蛋白的表达。获得的单克隆抗体和多克隆抗体是特异性的且具有高反应性。这些生物制剂的可用性可能有助于诊断分析和/或新的重组蛋白疫苗的开发。结论L21前导序列增强了昆虫细胞中产生的无毒C末端重组CPA蛋白的表达。获得的单克隆抗体和多克隆抗体是特异性的且具有高反应性。这些生物制剂的可用性可能有助于诊断分析和/或新的重组蛋白疫苗的开发。结论L21前导序列增强了昆虫细胞中产生的无毒C末端重组CPA蛋白的表达。获得的单克隆抗体和多克隆抗体是特异性的且具有高反应性。这些生物制剂的可用性可能有助于诊断分析和/或新的重组蛋白疫苗的开发。
更新日期:2020-04-22
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