金黄色葡萄球菌肠毒素B突变体（N23S,H32N和K172E）的构建及其对肉鸡免疫佐剂作用的研究
发布时间：2018-10-05 07:31
【摘要】：金黄色葡萄球菌肠毒素B(SEB)是由金黄色葡萄球菌分泌产生的一种外毒素,也是一种超抗原。SEB能够非特异性地激活免疫细胞,尤其是T细胞。产生众多细胞因子,能够提高特异性抗原的免疫原性。因而SEB具备成为免疫佐剂的潜力。但由于SEB对人和哺乳动物具有较大的毒副作用,因此其佐剂活性在畜牧兽医领域的应用受到限制。为了减轻或消除SEB的毒性作用,我们通过PCR定点突变技术,构建SEB突变体(N23S,H32N和K172E),筛选出超抗原活性较好的突变体。同时研究SEB及其突变体在肉鸡体内的安全性及其佐剂活性,为促进SEB作为免疫佐剂在兽医领域的应用奠定基础。本试验主要包括三个部分工作:1.获得SEB突变重组蛋白利用PCR方法对SEB的3个关键抗原位点进行突变,包括N23S,H32N和K172E,并构建了原核表达质粒p ET-28a-SEB,p ET-28a-SEBM23(N23S),p ET-28a-SEBM32(H32N),p ET-28a-SEBM172(K172E),p ET-28a-SEBM23/172(N23S+K172E),p ET-28a-SEBM32/172(H32N+K172E),p ET-28a-SEBM23/32/172(N23S+H32N+K172E)。在大肠杆菌中诱导表达并纯化获得7种重组蛋白。2.SEB及其突变体SEBM32/172对AIV-H9/NDV二联灭活苗的佐剂作用分别用重组蛋白SEB和SEBM32/172配合H9亚型禽流感、新城疫二联灭活苗免疫肉鸡,通过HI实验和MTT实验评价SEB及其突变体SEBM32/172对灭活疫苗的佐剂作用。结果显示在免疫后第14-28天5μg SEBM32/172能不同程度的提高灭活疫苗中AIV-H9的免疫效果,对NDV的作用不明显;1μg或10μg SEBM32/172和SEB的对灭活疫苗的佐剂效应不稳定。淋巴细胞活性检测结果显示1μg SEB和5μg SEBM32/172在免疫后第7天显著高于疫苗对照组,其他时间段存在一定差异。3.SEB及其突变体SEBM32/172在肉鸡体内的安全性评价为了研究突变SEBM32/172后能否降低SEB对机体毒性作用,我们用10倍佐剂剂量(50μg/只)的重组蛋白SEB和SEBM32/172分别肌肉注射肉鸡,分别在接种后第7、14、21天进行剖检取样制备组织病理切片,进行病理学观察。在试验过程中肉鸡表现正常,脏器没有明显的肉眼可见的组织病变,但在显微镜下,实验组肉鸡的肾脏存在不同程度的病理损伤,如肾小管变性坏死、肾小球肿大、肾小球肾炎等,病理损伤程度随时间的增加而加重,而SEBM32/172组的病理损伤比SEB组的病理损伤程度低,表明双突变SEBH32NK172E能降低SEB对肉鸡的毒性作用。
[Abstract]:Staphylococcus aureus enterotoxin B (SEB) is an exotoxin secreted by Staphylococcus aureus. It is also a superantigen. SEB can activate immune cells, especially T cells. The production of many cytokines can improve the immunogenicity of specific antigens. Therefore, SEB has the potential to become an immune adjuvant. However, the application of SEB adjuvant in animal husbandry and veterinary field is limited because of its toxicity and side effects to human and mammal. In order to reduce or eliminate the toxicity of SEB, we constructed SEB mutants (N23SnH32N and K172E) by PCR site-directed mutagenesis, and screened out the mutants with better superantigen activity. At the same time, the safety and adjuvant activity of SEB and its mutants in broilers were studied, which laid a foundation for the application of SEB as immune adjuvant in veterinary field. The experiment consists of three parts: 1. 鑾峰緱SEB绐佸彉閲嶇粍铔嬬櫧鍒╃敤PCR鏂规硶瀵筍EB鐨，
本文编号：2252533
[Abstract]:Staphylococcus aureus enterotoxin B (SEB) is an exotoxin secreted by Staphylococcus aureus. It is also a superantigen. SEB can activate immune cells, especially T cells. The production of many cytokines can improve the immunogenicity of specific antigens. Therefore, SEB has the potential to become an immune adjuvant. However, the application of SEB adjuvant in animal husbandry and veterinary field is limited because of its toxicity and side effects to human and mammal. In order to reduce or eliminate the toxicity of SEB, we constructed SEB mutants (N23SnH32N and K172E) by PCR site-directed mutagenesis, and screened out the mutants with better superantigen activity. At the same time, the safety and adjuvant activity of SEB and its mutants in broilers were studied, which laid a foundation for the application of SEB as immune adjuvant in veterinary field. The experiment consists of three parts: 1. 鑾峰緱SEB绐佸彉閲嶇粍铔嬬櫧鍒╃敤PCR鏂规硶瀵筍EB鐨，
本文编号：2252533
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