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

Abstract

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

CLC Number:

S855.3

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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Optimized Expression of Fowl Adenovirus Serotype 4 and 8b Tandem Truncated Fiber2-Fiber Protein in Pichia pastoris

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