Sari Agriculture Science and Natural Resource University (SANRU), Genetics and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari, Iran.
Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Pajoohesh BLVD, Tehran-Karaj HWY, km 15, Tehran 1497716316, Iran.
Vaccine. 2020 Oct 27;38(46):7284-7291. doi: 10.1016/j.vaccine.2020.09.047. Epub 2020 Oct 2.
Following the ban on the use of in-feed antimicrobials, necrotic enteritis (NE) NE is the most important clostridial disease. Vaccination has been considered as a possible approach to prevent NE. Our previous study showed that a chimeric protein product consisting of antigenic epitopes of NetB, Alpha-toxin and Zinc metallopeptidase (Zmp) triggered immune response against C. perfringens. In the current study we optimized the chimeric gene and constructed a fusion protein containing NetB, Alpha-toxin and Metallopeptidase (NAM) for expressing in tobacco plant to use as an edible vaccine for immunizing the chicken against NE. Simultaneously, we expressed and purified a His-tagged recombinant version of the NAM (rNAM) expressed in E. coli BL21 for subcutaneous immunization of chickens. Immunized birds produced strong humoral immune responses against both edible plant-based and parenteral purified rNAM. The responses were determined by the mean titer of antibody in blood samples to be around 9000 and 32,000, for edible and injected rNAM, respectively. Birds immunized subcutaneously showed the most striking responses. However the edible vaccine provided a more long lasting IgY response 14 days after the third vaccination compared to the injected birds. Chickens immunized with either lyophilized leaves expressing rNAM or purified rNAM, subsequently were subjected to the challenge with a virulent C. perfringens strain using an NE disease model. Our results showed that birds immunized both parenterally and orally with recombinant chimeric vaccine were significantly protected against the severity of lesion in the intestinal tract, but the protection provided with the injectable form of the antigen was greater than that of the oral form. Further analysis is needed to check whether these strategies can be used as the potential platform for developing an efficient vaccine against NE.
在禁止饲料中使用抗生素后，坏死性肠炎（NE）是最重要的梭菌病。疫苗接种已被认为是预防 NE 的一种可能方法。我们之前的研究表明，由 NetB、α-毒素和锌金属肽酶（Zmp）的抗原表位组成的嵌合蛋白产品引发了针对产气荚膜梭菌的免疫反应。在本研究中，我们优化了嵌合基因，并构建了一种含有 NetB、α-毒素和金属肽酶（NAM）的融合蛋白，在烟草植物中表达，用作免疫鸡预防 NE 的可食用疫苗。同时，我们在大肠杆菌 BL21 中表达并纯化了一个带有 His 标签的 NAM 重组版本（rNAM），用于鸡的皮下免疫。免疫鸡产生了针对可食用植物源性和原代纯化 rNAM 的强烈体液免疫反应。通过血液样本中抗体的平均效价来确定反应，可食用和注射 rNAM 的效价分别约为 9000 和 32000。皮下免疫的鸟类表现出最显著的反应。然而，与注射组相比，第三次免疫后 14 天，可食用疫苗提供了更持久的 IgY 反应。用表达 rNAM 的冻干叶片或纯化的 rNAM 免疫的鸡随后用一种强毒产气荚膜梭菌菌株进行 NE 疾病模型挑战。我们的结果表明，用重组嵌合疫苗进行全身和口服免疫的鸡在肠道病变的严重程度上受到显著保护，但注射形式的抗原提供的保护大于口服形式。需要进一步分析以检查这些策略是否可用于开发针对 NE 的有效疫苗的潜在平台。
