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

摘要/Abstract

摘要： 【目的】在毕赤酵母中构建猫血清白蛋白（feline serum albumin，FSA）表达系统，并探索其最适的基本表达条件，为FSA的生产提供一种新方法。【方法】在GenBank上获取FSA碱基序列，进行密码子优化并合成，将优化的密码子构建到载体pPIC9K上，经PCR和双酶切验证后通过电击转化将重组质粒FSA-pPIC9K转化毕赤酵母GS115，依次经MD平板和G418筛选后进行菌落PCR获得阳性菌株，随机选取阳性菌株经甲醇诱导表达96 h后，取表达上清液进行Western blotting验证。选取表达量较高的菌株分别在不同温度（24、26、28和30 ℃）、pH （4.0、5.0、6.0、7.0和8.0）和甲醇诱导量（其中1组为每24 h添加0.5%，其余4组为每12 h分别添加0.5%、1.0%、1.5%和2.0%）的条件下诱导表达96 h，取表达上清液进行Western blotting验证。【结果】菌落PCR结果显示，获得2条大小分别约为2.3和2.2 kb的目的基因条带和毕赤酵母AOX1基因扩增条带，诱导表达96 h后取表达上清液进行Western blotting验证，在70 ku左右出现特异性条带。通过基本条件优化发现该蛋白在28 ℃，pH为6.0且每12 h添加0.5%的甲醇条件下表达量较高。【结论】毕赤酵母GS115可稳定表达FSA。

关键词: 猫血清白蛋白(FSA); 毕赤酵母; 分泌表达

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

中图分类号:

赵明明, 李星颖, 江文康, 樊全宝, 赖健仪, 何诗, 何静, 白银山, 刘璨颖, 陈胜锋, 陈志胜, 张晖, 王丙云. 猫血清白蛋白在毕赤酵母中的分泌表达[J]. 中国畜牧兽医, 2022, 49(3): 897-903.

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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