三种禽病毒抗原的串联表达、纯化及活性鉴定

Co-expression,purification and bioassay of three avian viral antigens

为实现多个基因在同一菌株中均一可溶性表达,简化基因工程亚单位多联多价疫苗中抗原生产的工艺步骤,本研究选用Ⅰ群4型禽腺病毒(FAdV-4)Fiber-2蛋白、鸡传染性法氏囊病病毒(IBDV)VP2蛋白和减蛋综合征病毒(EDSV)Fiber蛋白3种来自不同禽病毒的抗原为研究对象,利用原核表达系统,通过密码子优化、载体启动子改造和基因串联顺序优化,获得单一载体/多重转录单元的共表达重组质粒。将共表达重组质粒转化大肠杆菌BL21(DE3)菌株,进行3个基因的共表达。纯化后的蛋白进行Western blotting和蛋白活性检测。结果表明,目的基因经过密码子优化、载体启动子改造和基因串联顺序的优化后,获得均一可溶性共表达的3种蛋白,纯化后蛋白纯度大于80%,Western blotting分析和琼脂扩散试验表明串联表达的3种蛋白具有免疫反应性和抗原活性。文中通过目的基因密码子优化、表达载体启动子改造和基因串联等关键技术的突破,首次实现了3种不同禽病毒抗原的高效、均一、可溶性串联表达和纯化,为基因工程亚单位多联多价疫苗的研制奠定了基础。

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain,and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine,we co-expressed three avian virus proteins including the fowl adenovirus serotype 4(FAdV-4)Fiber-2 protein,infectious bursal disease virus(IBDV)VP2 protein and egg-drop syndrome virus(EDSV)Fiber protein in E.coli BL21(DE3)cells after optimization of gene codon,promoter,and tandem expression order.The purified proteins were analyzed by Western blotting and agar gel precipitation(AGP).The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%.Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity.It is the first time to achieve the three different avian virus antigens co-expression and co-purification,which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.