中国明对虾ALFFc基因在毕赤酵母中的表达

doi:10.3969/j.issn.1000-1565.2018.01.010

河北大学学报(自然科学版) ›› 2018, Vol. 38 ›› Issue (1): 59-64.DOI: 10.3969/j.issn.1000-1565.2018.01.010

中国明对虾ALFFc基因在毕赤酵母中的表达

唐婷,陆美柯,马新建,李婷,柳峰松

收稿日期:2017-05-26 出版日期:2018-01-25 发布日期:2018-01-25
通讯作者: 柳峰松(1976-),男,河北任丘人,河北大学教授,主要从事无脊椎动物分子免疫学研究.E-mail:liufengsong@hbu.edu.cn
作者简介:唐婷(1982—),女,广西来宾人,河北大学副教授,主要从事无脊椎动物分子免疫学研究. E-mail:tangting@hbu.edu.cn
基金资助:
国家自然科学基金资助项目(31572327);河北省自然科学基金资助项目(C2015201013);河北大学杰出青年基金资助项目(2015JQ05)

Expression of Chinese shrimp Fenneropenaeus chinensis ALFFc in Pichia pastoris

TANG Ting, LU Meike, MA Xinjian, LI Ting, LIU Fengsong

Key Laboratory of Zoological Systematics and Application, College of Life Sciences, Hebei University, Baoding 071002, China

Received:2017-05-26 Online:2018-01-25 Published:2018-01-25

摘要/Abstract

摘要： 抗脂多糖因子(ALF)是一种抗菌活性小分子蛋白,在机体免疫系统中发挥重要作用,被认为具有替代抗生素药物的前景.该研究在已有中国明对虾ALFFc原核表达载体pET-DsbA-ALFFc的基础上,通过PCR在ALFFc的5’和3’端加上酶切位点,扩增到的片段与表达载体pPIC9K连接构建重组表达载体pPIC9K-ALFFc,转化到毕赤酵母GS115细胞中,通过筛选及鉴定获得甲醇利用型酵母转化子.通过甲醇诱导表达并镍柱纯化获得rALFFc,经SDS-PAGE和Western blot分析证明ALFFc在毕赤酵母中成功表达.

关键词: 中国明对虾, 抗脂多糖因子(ALF), 毕赤酵母, 重组表达

Abstract: Anti-lipopolysaccharide factor(ALF)is a low molecular mass protein with antimicrobial activities and plays an important role in immune response against microbial invasion.Thus, they are considered to be good substitutes for traditional antibiotics.In the present study, the prokaryotic recombinant expression vector “pET-DsbA-ALFFc” was used as the template of PCR, which was constructed in previous study.EcoRI and NotI restriction sites were added to 5’ and 3’ ends of the ALFFc.The ALFFc fragment was ligated to pPIC9K vector to construct a recombinant expression vector pPIC9K-ALFFc, which then was transformed into competent Pichia pastoris GS115.Recombinant ALFFc(rALFFc)was induced with methanol and purified by Ni-chelating affinity chromatography.SDS-PAGE and Western blot analysis indicated that the rALFFc was expressed successfully in P.pastoris.

Key words: Fenneropenaeus chinensis, anti-lipopolysaccharide factor(ALF), Pichia pastoris, recombinant expression

中图分类号:

唐婷,陆美柯,马新建,李婷,柳峰松. 中国明对虾ALFFc基因在毕赤酵母中的表达[J]. 河北大学学报(自然科学版), 2018, 38(1): 59-64.

TANG Ting, LU Meike, MA Xinjian, LI Ting, LIU Fengsong. Expression of Chinese shrimp Fenneropenaeus chinensis ALFFc in Pichia pastoris[J]. Journal of Hebei University (Natural Science Edition), 2018, 38(1): 59-64.

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中国明对虾ALFFc基因在毕赤酵母中的表达

Expression of Chinese shrimp Fenneropenaeus chinensis ALFFc in Pichia pastoris

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