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.

在禁止使用饲料中的抗菌剂之后，坏死性肠炎（NE）NE是最重要的梭菌病。接种疫苗被认为是预防NE的一种可能方法。我们先前的研究表明，由NetB抗原表位，α-毒素和Zinc Metallopeptidase（Zmp）组成的嵌合蛋白产品可触发针对产气荚膜梭菌的免疫反应。在当前的研究中，我们优化了嵌合基因，并构建了一种包含NetB，α毒素和金属肽酶（NAM）的融合蛋白，可在烟草植物中表达，用作可食用的疫苗，用于免疫鸡的NE。同时，我们表达并纯化了在大肠杆菌中表达的NAM（rNAM）的His标记重组形式。BL21用于鸡的皮下免疫。免疫的禽类对基于食用植物的和肠胃外纯化的rNAM产生强烈的体液免疫反应。

对于可食用和注射的rNAM，通过血样中抗体的平均滴度确定响应，分别约为9000和32,000。皮下免疫的鸟类表现出最惊人的反应。然而，与注射的禽类相比，可食用疫苗在第三次疫苗接种后14天提供了更持久的IgY反应。用表达rNAM的冻干叶或纯化rNAM免疫的鸡免疫后，用产气荚膜梭状芽胞杆菌进行攻击使用NE疾病模型。我们的研究结果表明，用重组嵌合疫苗进行肠胃外和口服免疫的禽类可以显着保护肠道免受病变的严重程度，但是抗原注射剂提供的保护作用大于口服形式。需要进一步分析，以检查这些策略是否可以用作开发针对NE的有效疫苗的潜在平台。