联合策略优化犬α干扰素的酵母表达<sup>*</sup>

doi:10.13523/j.cb.2107010

中国生物工程杂志

2022, Vol. 42

Issue (1/2): 88-95 DOI: 10.13523/j.cb.2107010

技术与方法

联合策略优化犬α干扰素的酵母表达^*

李丁^1,^2,³,李兰^1,^2,³,安允飞⁴,毕振威⁵,于晓明^1,^2,³,陈瑾^1,^2,³,郑其升^1,^2,^3,^**(

)

1 江苏省农业科学院动物免疫工程研究所南京 210014
2 江苏省动物重要疫病与人兽共患病防控协同创新中心扬州 225009
3 江苏省食品质量安全重点实验室-省部共建国家重点实验室培育基地南京 210014
4 南京农业大学食品科技学院南京 210095
5 江苏省农业科学院兽医研究所南京 210014

Optimized Yeast Expression of Canine Interferon-α Using a Combined Strategy

LI Ding^1,^2,³,LI Lan^1,^2,³,AN Yun-fei⁴,BI Zhen-wei⁵,YU Xiao-ming^1,^2,³,CHEN Jin^1,^2,³,ZHENG Qi-sheng^1,^2,^3,^**(

)

1 Institute of Veterinary Immunology & Engineering, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
2 Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou 225009, China
3 Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base, Ministry of Science and Technology, Nanjing 210014, China
4 College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China
5 Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China

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摘要：

前期研究在毕赤酵母中分泌表达了犬α干扰素(CaIFN),其活性较高但产量未达到理想水平。目前工作的重点是提高重组蛋白产量。构建含有1、2、4、6、8拷贝CaIFN基因的酵母转化子,其中KM-6CaIFN产量最高,与KM-1CaIFN相比提高了200.6%。共表达8种分子伴侣,其中Hac蛋白能够提高KM-6CaIFN和KM-8CaIFN的目的蛋白产量,提高幅度分别为32.1%和113.1%。在此基础上继续增加CaIFN拷贝数,可观察到KM-12CaIFN-Hac的产量最高,是KM-8CaIFN-Hac的1.33倍,同时是KM-1CaIFN的5.61倍。通过与梯度稀释的BSA进行蛋白质条带比较,评估KM-12CaIFN-Hac的靶蛋白产量约为581 mg/L,为目前报道中酵母产犬α干扰素最高值。

关键词： 犬α干扰素; 毕赤酵母; 重组蛋白产量; 多拷贝; 分子伴侣

Abstract:

Canine interferon-α(CaIFN) was expressed in Pichia pastoris in the previous studies, which had high biological activity and relatively low yield. Therefore, increasing the yield of recombinant protein is the key task to promote the application of CaIFN. Yeast transformants containing 1, 2, 4, 6, and 8 copies of CaIFN gene were generated, and the yield of KM-6CaIFN was the highest, which was 200.6% higher than that of KM-1CaIFN. 8 molecular chaperones were co-expressed with CaIFN, and the Hac protein could increase the target protein yield of KM-6CaIFN and KM-8CaIFN by 32.1% and 113.1%, respectively. More copies of CaIFN were integrated into KM-8CaIFN-Hac strain. SDS-PAGE analysis showed that the yield of KM-12CaIFN-Hac was 1.33 times that of KM-8CaIFN-Hac, and 5.61 times that of KM-1CaIFN, respectively. By comparing protein bands with serially dilution BSA, the yield of KM-12CaIFN-Hac was estimated to be about 581 mg/L, which is the highest value in P. pastoris reported so far.

Key words: CaIFN Pichia pastoris Recombinant protein yield Multi-copy Molecular chaperones

收稿日期: 2021-07-03 出版日期: 2022-03-03

ZTFLH:

Q819

基金资助: * 国家自然科学基金(32002316);国家自然科学基金(31802220);江苏省农业自主创新专项资助项目(CX(20)3096)

通讯作者: 郑其升 E-mail: njcvc1302@163.com

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引用本文:

李丁,李兰,安允飞,毕振威,于晓明,陈瑾,郑其升. 联合策略优化犬α干扰素的酵母表达^*[J]. 中国生物工程杂志, 2022, 42(1/2): 88-95.

LI Ding,LI Lan,AN Yun-fei,BI Zhen-wei,YU Xiao-ming,CHEN Jin,ZHENG Qi-sheng. Optimized Yeast Expression of Canine Interferon-α Using a Combined Strategy. China Biotechnology, 2022, 42(1/2): 88-95.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2107010 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I1/2/88

表1 qPCR所用引物

表2 共表达的分子伴侣

图1 KM-1CaIFN发酵液上清的SDS-PAGE (a)和Western blot(b) 检测

图2 双酶切验证重组表达质粒

表3 酵母转化子的拷贝数分析

图3 基因拷贝数对CaIFN产量的影响

图4 共表达分子伴侣对KM-6CaIFN菌株产CaIFN的影响

图5 共表达分子伴侣对KM-8CaIFN菌株产CaIFN的影响

图6 增加基因拷贝数对共表达菌株产CaIFN的影响

图7 KM-1CaIFN、KM-12CaIFN-Hac发酵液上清的CaIFN蛋白定量

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