首页>
外文期刊>BMC Biotechnology
>Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies
【24h】
Generation of recombinant baculovirus expressing atoxic C-terminal CPA toxin of Clostridium perfringens and production of specific antibodies
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. 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. 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.
展开▼
机译：Clostridium perfringens是动物和人类中几种疾病和肠道感染的致病因子。 C. proFringens的毒力很大程度上是毒素的毒素;其中，α毒素（CPA）在组毒毒性感染（气坏疽）中起着至关重要的作用。 CPA毒素由两个结构域组成，即磷脂酶C活性位点，其位于N-末端结构域氨基酸（AA残基1-250），以及负责的C末端区域（AA残基251-370）毒素与膜磷脂在钙离子存在下的相互作用。所有目前生产的梭菌疫苗含有衍生自营养的培养上清液的类毒素，主要是使用福尔马林。 CPA是一种免疫原性抗原;最近，已经表明，用大肠杆菌中产生的毒素的C末端结构域免疫的小鼠免受C.流细管感染和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制备，而不是抗截短的形式。在本研究中，我们首次报道了;关于产生能够产生缺少缺失的RCPA毒素（RBACCPA250-363H6）的重组杆状病毒缺乏缺失的N-末端结构域和推定信号序列的28个氨基酸（AA）。在平移起始密码子ATG上游的L21共有序列的插入急剧增加了基于杆状病毒的表达系统中重组蛋白的产率。通过Ni-NTA亲和层析纯化蛋白质，在CaCO-2细胞中证实了体外缺乏毒性。产生多克隆抗体和8种杂交瘤分泌单克隆抗体并测试以评估特异性和反应性。抗血清，抗片段RBACCPA250-3636中和全长PLC的磷脂酶C活性。 L21前导序列增强了在昆虫细胞中产生的毒性C末端重组CPA蛋白的表达。获得的单克隆和多克隆抗体是特异性且高反应性的。这些生物学的可用性可以有助于诊断测定和/或新的重组蛋白质疫苗的发展。
展开▼
