猫血清白蛋白在毕赤酵母中的分泌表达

doi:10.16431/j.cnki.1671-7236.2022.03.011

Abstract

Abstract: 【Objective】 The purpose of this study was to construct the expression system of feline serum albumin (FSA) in Pichia pastoris, and to explore its optimal basic expression conditions, so as to provide a new method for the production of FSA.【Method】 The FSA base sequence was obtained from GenBank, and the codon was optimized and synthesized.The optimized codon was constructed into vector pPIC9K.After PCR and double enzyme digestion verification, the recombinant plasmid FSA-pPIC9K was transformed into Pichia pastoris GS115 by electric shock transformation.The positive strains were obtained by colony PCR after screening by MD plate and G418.The positive strains were randomly selected and induced by methanol for 96 h, the expression supernatant was taken for Western blotting verification.The strains with high expression were selected to induce expression for 96 h at different temperatures (24, 26, 28 and 30 ℃), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and methanol induction (0.5% was added every 24 h in one group and 0.5%, 1.0%, 1.5% and 2.0% were added every 12 h in the other four groups).The expression supernatant was taken for Western blotting verification.【Results】 The colony PCR results showed that two bands with the size of about 2.3 and 2.2 kb of target gene and Pichia pastoris AOX1 gene were obtained.After 96 h of induced expression, the expression supernatant was taken for Western blotting verification, and the specific bands appeared at about 70 ku.Through the optimization of basic conditions, it was found that the protein was highly expressed at 28 ℃, pH 6.0 and 0.5% methanol was added every 12 h.【Conclusion】 Pichia pastoris GS115 could stably express FSA.

Key words: feline serum albumin (FSA); Pichia pastoris; secretion expression

CLC Number:

ZHAO Mingming, LI Xingying, JIANG Wenkang, FAN Quanbao, LAI Jianyi, HE Shi, HE Jing, BAI Yinshan, LIU Canying, CHEN Shengfeng, CHEN Zhisheng, ZHANG Hui, WANG Bingyun. Secretory Expression of Feline Serum Albumin in Pichia pastoris[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 897-903.

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Secretory Expression of Feline Serum Albumin in Pichia pastoris

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