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禽腺病毒血清4型Fiber-2蛋白在毕赤酵母中的优化表达及其免疫原性分析
袁枫^1,2,宋慧琦²,侯磊^3,4,韦莉²,朱珊珊²,全荣²,王菁²,王丹²,姜海军²,刘浩^1,刘爵^3,4
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(1. 天津科技大学生物工程学院, 天津 300222;2. 北京市农林科学院畜牧兽医研究所, 北京 100097;3. 扬州大学兽医学院, 江苏扬州 225009;4. 扬州大学江苏省重大动物传染病与人畜共患病防控合作创新中心, 江苏扬州 225009)

摘要:

为研制禽腺病毒血清4型(Fowl adenovirus serotype 4，FAdV-4)亚单位疫苗，本研究对FAdV-4的Fiber-2蛋白在毕赤酵母中的发酵工艺进行了优化。首先根据酵母密码子偏好性优化Fiber-2基因，并且构建了重组真核表达载体pPIC9K-Fiber-2。将该质粒转化至毕赤酵母GS115菌株，经不同质量浓度G418筛选获得高拷贝重组菌。随后在摇瓶中进行诱导发酵时温度、时间和甲醇浓度的优化。用优化后的条件进行发酵表达并将上清液通过镍柱纯化得到了重组的Fiber-2蛋白。最后将蛋白与ISA-201-VG油佐剂混合并免疫21日龄SPF鸡进行免疫保护性试验。结果表明:在毕赤酵母中成功且高效的分泌表达了Fiber-2蛋白，分子量约为60 ku，在摇瓶发酵中诱导的最佳甲醇浓度、时间和温度分别为1.0%、72 h和30 ℃。优化后发酵液上清中的总蛋白量约为50 mg/L，重组Fiber-2蛋白达到8.7 mg/L。SDS -PAGE和Western blot结果表明采用镍柱对蛋白进行纯化可得到条带单一且具有良好免疫原性的重组Fiber-2蛋白。免疫保护性试验显示，由优化后的毕赤酵母表达Fiber-2蛋白制备的亚单位疫苗免疫雏鸡后，可以诱导较高的抗体水平并提供高达100%的超强FAdV-4毒株(GD616)的攻击保护。本研究为进一步采用毕赤酵母表达的重组Fiber-2蛋白制备FAdV-4亚单位疫苗的研发奠定了基础。

关键词: 禽腺病毒血清4-型 Fiber-2蛋白毕赤酵母亚单位疫苗

DOI：10.11841/j.issn.1007-4333.2022.02.13

投稿时间：2021-03-26

基金项目:国家重点研发计划(2016YFD0500806)；国家肉鸡产业技术体系岗位科学家项目(CARS-41-G14)；江苏省高等学校重点学科发展计划(PAPD)

Optimized expression of fowl adenovirus type 4 Fiber-2 protein in Pichia pastoris and immunogenicity analysis of expressed product

YUAN Feng^1,2,SONG Huiqi²,HOU Lei^3,4,WEI Li²,ZHU Shanshan²,QUAN Rong²,WANG Jing²,WANG Dan²,JIANG Haijun²,LIU Hao^1*,LIU Jue^3,4*

(1.School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300222, China;2.Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China;3.College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;4.Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China)

Abstract:

In order to develop the fowl adenovirus serotype 4(FAdV-4)subunit vaccine, the fermentation process of the Fiber-2 protein of FAdV-4 in Pichia pastoris was optimized. Firstly, the Fiber-2 gene was optimized according to the codon preference of yeast, and the recombinant eukaryotic expression vector pPIC9K-Fiber-2 was constructed. The plasmid was transformed into GS115 strain and the high copy recombinant strain was obtained by screening with different concentrations of G418. Then the temperature, time and methanol concentration of induced fermentation were optimized in a shaking flask. The recombinant Fiber-2 protein was obtained by fermentation under the optimized conditions and the supernatant was purified by nickel column. Finally, the protein was mixed with ISA-201-VG oil adjuvant and 21-day-old SPF chickens were immunized for the immune protection test. The results showed that Fiber-2 protein was successfully secreted and efficiently expressed in Pichia pastoris, and its molecular weight was about 60 ku. The optimal methanol concentration, time and temperature in shake flask fermentation were 1. 0%(v/v), 72 h, and 30 ℃, respectively. After optimization, the total protein in the supernatant of the fermentation broth was about 50 mg/L, and the recombinant Fiber-2 protein reached 8. 7 mg/L. SDS-PAGE and Western blot showed that the recombinant Fiber-2 protein with a single band and good reactivity could be obtained by purifying the protein with a nickel column. Immune protective tests showed that the subunits vaccine prepared by the optimized optimized P. pastoris expressing Fiber-2 protein could induce high antibody levels and provide up to 100% attack protection against hypervirulent FAdV-4(GD616). This study laid a foundation for the research and development of FAdV-4 subunit vaccine using recombinant Fiber-2 protein expressed in P. pastoris.

Key words: fowl adenovirus serotype 4 Fiber-2 protein Pichia pastoris subunit vaccine