鸭腺病毒3型Fiber-2蛋白在酵母细胞中的表达及免疫原性研究

Expression and immunogenicity of Duck adenovirus 3 Fiber-2 protein in yeast cells

[目的]利用马克斯克鲁维酵母(Kluyveromyces marxianus)系统高效表达鸭腺病毒3型(DAdV-3)Fiber-2蛋白,用于制备安全、高效、低成本的鸭腺病毒亚单位疫苗。[方法]以食品安全级的马克斯克鲁维酵母为宿主,对DAdV-3 Fiber-2蛋白进行重组表达,建立高密度发酵和纯化工艺,获得目的蛋白并制备成亚单位疫苗,然后进行动物试验。试验分为低剂量组(琼扩效价1∶64,每羽34μg)、高剂量组(1∶128,每羽68μg)、空白组和攻毒对照组,在免疫后第10天和第21天采血检测血清中的抗体水平,攻毒7 d后采肛拭子检测排毒量;通过抗体水平和排毒量评估该亚单位疫苗的保护效果。[结果]通过SDS-PAGE、Western blot和琼脂糖凝胶扩散(AGP)试验验证fiber-2基因在酵母中成功表达。重组菌经高密度发酵,目的蛋白纯化后检测其表达水平,琼扩效价可达1∶128。动物试验结果显示,疫苗免疫组在免疫后第21天所测得血清中的抗体水平显著高于空白组;疫苗免疫组在攻毒7 d后的排毒量显著低于攻毒对照组。[结论]DAdV-3 Fiber-2蛋白能在马克斯克鲁维酵母细胞中进行高效表达,并且用该蛋白制备的亚单位疫苗对鸭3型腺病毒具有一定的保护作用。

[Objectives]Duck adenovirus 3(DAdV-3)Fiber-2 protein was highly expressed in Kluyveromyces marxianus system to prepare a safe, efficient and low-cost duck adenovirus subunit vaccine. [Methods]The recombinant expression of DAdV-3 Fiber-2 protein was carried out with food safety grade K.marxianus as the host. High density fermentation and purification process were established to obtain the target protein, prepare subunit vaccine, and then carry out animal test. The test was divided into low dose group(which agarose gel diffusion(AGP)titer was 1∶64 and the antigen dose was 34 μg per duck),high dose group(which AGP titer was 1∶128 and the antigen dose was 68 μg per duck),blank control group and challenge control group, muscovy duck serum was taken to detect the antibody level on the 10th and 21st day after immunization, and anal swabs were taken to detect the detoxification amount 7 days after challenge. The protective effect of the subunit vaccine was evaluated by antibody level and detoxification amount. [Results]The expression of fiber-2 gene fragment in yeast was confirmed by SDS-PAGE,Western blot and AGP. The AGP titer of the target protein was 1∶128 after high-density fermentation and purification. The results of animal experiment showed that the antibody level of muscovy ducks in the immune group on the 21st day was significantly higher than that in the blank group;The amount of detoxification in the vaccine immunized group after 7 days was significantly lower than that in the challenge control group. [Conclusions]DAdV-3 Fiber-2 protein can be highly expressed in K.marxianus cells, and the vaccine has a certain protective effect on Duck adenovirus 3.