禽腺病毒血清4型和8b型串联截短Fiber2-Fiber蛋白在毕赤酵母中的优化表达

doi:10.16431/j.cnki.1671-7236.2023.08.003

摘要/Abstract

摘要： 【目的】在毕赤酵母中构建禽腺病毒(FAdV)血清4型和8b型串联截短Fiber2-Fiber蛋白表达系统,并探索适宜的表达条件。【方法】以前期构建的重组质粒pCold-Fiber2-Fiber为模板,用PCR法扩增Fiber2-Fiber片段,连接pPICZαA载体。经双酶切和测序鉴定后的阳性重组质粒pPICZαA-Fiber2-Fiber电转化毕赤酵母GS115。经MD平板和不同浓度Zeocin抗性筛选后进行PCR扩增和测序鉴定得到阳性菌株;用甲醇诱导表达阳性菌株,72 h后收集蛋白上清进行SDS-PAGE和液相色谱串联质谱(LC-MS/MS)鉴定。采用单因素变量法对不同pH(5.0、6.0、7.0和8.0)、甲醇浓度(0.5%、1.0%、1.5%和2.0%)、时间(24、48、72和96 h)进行表达条件优化,用SDS-PAGE鉴定,并用间接ELISA和Western blotting验证重组蛋白的反应原性。【结果】成功构建了重组质粒pPICZαA-Fiber2-Fiber,对表达72 h后的上清进行SDS-PAGE发现1条约70 ku的特异条带,经LC-MS/MS鉴定为目的蛋白。串联截短Fiber2-Fiber蛋白的最优诱导条件为pH 6.0、1%甲醇、诱导时间72 h。间接ELISA和Western blotting结果显示该重组蛋白能与FAdV-4和FAdV-8b阳性血清特异性结合。【结论】Fiber2-Fiber串联截短蛋白可在毕赤酵母中稳定表达并具有良好的反应原性,为禽腺病毒双价亚单位疫苗的研发奠定基础。

关键词: 禽腺病毒(FAdV); Fiber; 毕赤酵母; 分泌表达

Abstract: 【Objective】 The purpose of this study was to construct expression system of Fowl adenovirus (FAdV) serotype 4 and 8b tandem truncated Fiber2-Fiber protein in Pichia pastoris,and explore appropriate expression conditions.【Method】 Using the previously constructed recombinant plasmid pCold-Fiber2-Fiber as the template,the Fiber2-Fiber fragments were amplified by PCR and the pPICZαA vector was connected.The positive recombinant plasmid by restriction digestion and sequencing was electrically transferred into Pichia pastoris GS115.After screening by MD plate and different concentrations of Zeocin resistance,the positive strains were identified by PCR amplification and sequencing.The positive strains were induced by methanol for 72 h,and the protein supernatant was collected for SDS-PAGE and liquid chromatography tandem mass spectrometry (LC-MS/MS) identification.Single factor variable test was used to optimize different expression conditions including pH (5.0,6.0,7.0 and 8.0),methanol concentration(0.5%,1.0%,1.5% and 2.0%) and time (24,48,72 and 96 h),and identified by SDS-PAGE.The reactogenicity of the recombinant protein was verified by indirect ELISA and Western blotting.【Result】 The recombinant plasmid pPICZαA-Fiber2-Fiber was successfully constructed.SDS-PAGE was performed on the supernatant expressed for 72 h and a specific band about 70 ku was found,which was identified as the target protein by LC-MS/MS.The optimum induction conditions of tandem truncated Fiber2-Fiber protein were pH 6.0,1% methanol and induction time 72 h.The results of indirect ELISA and Western blotting showed that the protein could specifically bind to the positive serum of FAdV-4 and FAdV-8b.【Conclusion】 The tandem truncated Fiber2-Fiber protein could be stably expressed in Pichia pastoris and had good reactivity,which laid a foundation for the development of FAdV bivalent subunit vaccine.

Key words: Fowl adenovirus (FAdV); Fiber; Pichia pastoris; secretory expression

中图分类号:

S855.3

迟丽丽, 刘健, 陈志远, 李树凡, 张玫瑜, 于泽海, 李丹, 尹燕博, 徐守振. 禽腺病毒血清4型和8b型串联截短Fiber2-Fiber蛋白在毕赤酵母中的优化表达[J]. 中国畜牧兽医, 2023, 50(8): 3056-3064.

CHI Lili, LIU Jian, CHEN Zhiyuan, LI Shufan, ZHANG Meiyu, YU Zehai, LI Dan, YIN Yanbo, XU Shouzhen. Optimized Expression of Fowl Adenovirus Serotype 4 and 8b Tandem Truncated Fiber2-Fiber Protein in Pichia pastoris[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(8): 3056-3064.

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