Shahid Naila, Samiullah Tahir Rehman, Shakoor Sana, Latif Ayesha, Yasmeen Aneela, Azam Saira, Shahid Ahmad Ali, Husnain Tayyab, Rao Abdul Qayyum
Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan.
Front Vet Sci. 2020 Aug 24;7:499. doi: 10.3389/fvets.2020.00499. eCollection 2020.
Newcastle disease (ND) is a viral disease that causes labored breathing, periorbital oedema, and ataxia in the majority of avian species. The available vaccines against Newcastle disease virus (NDV) are limited, owing to their low reactivity and multiple dosage requirements. Plant-based machinery provides an attractive and safe system for vaccine production. In the current study, we attempted to express fusion (F) and hemagglutinin-neuraminidase (HN) proteins (the protective antigens against NDV) under constitutive 35S and seed-specific Zein promoters, respectively. Almost 2-7.1-fold higher expression of F gene mRNA in transgenic corn leaves and 8-28-fold higher expression of HN gene mRNA in transgenic corn seeds were observed, when the expression was analyzed by real-time PCR on a relative basis as compared to non-transgenic control plant material (Leaves and seeds). Similarly, 1.66 μg/ml of F protein in corn leaves, i.e., 0.5% of total soluble protein, and 2.4 μg/ml of HN protein in corn seed, i.e., 0.8% of total seed protein, were found when calculated through ELISA. Similar levels of immunological response were generated in chicks immunized through injection of -produced pET F and pET HN protein as in chickens orally fed leaves and seeds of maize with expressed immunogenic protein. Moreover, the detection of anti-NDV antibodies in the sera of chickens that were fed maize with immunogenic protein, and the absence of these antibodies in chickens fed a normal diet, confirmed the specificity of the antibodies generated through feeding, and demonstrated the potential of utilizing plants for producing more vaccine doses, vaccine generation at higher levels and against other infectious diseases.
新城疫（ND）是一种病毒性疾病，可导致大多数禽类呼吸困难、眶周水肿和共济失调。现有的抗新城疫病毒（NDV）疫苗由于反应性低和需要多次接种而受到限制。基于植物的生产体系为疫苗生产提供了一个有吸引力且安全的系统。在本研究中，我们试图分别在组成型35S启动子和种子特异性玉米醇溶蛋白启动子下表达融合（F）蛋白和血凝素神经氨酸酶（HN）蛋白（抗NDV的保护性抗原）。与非转基因对照植物材料（叶片和种子）相比，通过实时PCR相对分析表达情况时，在转基因玉米叶片中观察到F基因mRNA表达量几乎高2 - 7.1倍，在转基因玉米种子中HN基因mRNA表达量高8 - 28倍。同样，通过酶联免疫吸附测定法计算发现，玉米叶片中F蛋白含量为1.66μg/ml，即占总可溶性蛋白的0.5%，玉米种子中HN蛋白含量为2.4μg/ml，即占种子总蛋白的0.8%。通过注射表达产生的pET F和pET HN蛋白免疫的雏鸡，与口服含有表达免疫原性蛋白的玉米叶片和种子的鸡产生的免疫反应水平相似。此外，在喂食含有免疫原性蛋白玉米的鸡血清中检测到抗NDV抗体，而喂食正常饲料的鸡血清中没有这些抗体，这证实了通过喂食产生的抗体的特异性，并证明了利用植物生产更多疫苗剂量、更高水平生产疫苗以及针对其他传染病生产疫苗的潜力。
