水貂白细胞介素-4基因的克隆、序列分析与原核表达

doi:10.16431/j.cnki.1671-7236.2017.01.004

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (1): 30-37.doi: 10.16431/j.cnki.1671-7236.2017.01.004

水貂白细胞介素-4基因的克隆、序列分析与原核表达

范思宁¹, 张海玲², 赵建军², 王洋², 胡博², 吕爽², 马凡舒², 李欣彤², 凌明玉¹, 赵辉¹, 廉士珍², 闫喜军²

1. 吉林农业大学动物科学技术学院, 长春 130118;
2. 中国农业科学院特产研究所, 长春 130112

收稿日期:2016-06-20 出版日期:2017-01-20 发布日期:2017-01-19
通讯作者: 闫喜军 E-mail:tcsyxj@126.com
作者简介:范思宁(1990-),女,吉林公主岭人,硕士生,研究方向:预防兽医学,E-mail:fansining@126.com
基金资助:
吉林省科技发展计划项目（20140520172JH）；吉林省科技发展计划项目（20140307002NY、20140204072NY）

Cloning,Sequence Analysis and Prokaryotic Expression of Mink Interleukin-4 Gene

FAN Si-ning¹, ZHANG Hai-ling², ZHAO Jian-jun², WANG Yang², HU Bo², LV Shuang², MA Fan-shu², LI Xin-tong², LING Ming-yu¹, ZHAO Hui¹, LIAN Shi-zhen², YAN Xi-jun²

1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;
2. Division of Infectious Diseases of Special Economic Animal, Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences, Changchun 130112, China

Received:2016-06-20 Online:2017-01-20 Published:2017-01-19

摘要/Abstract

摘要：

为了获得高纯度的白细胞介素-4（IL-4）蛋白，对分离得到的水貂外周血淋巴细胞经植物血凝素（PHA）诱导后，提取淋巴细胞总RNA，根据GenBank中登录的雪貂IL-4基因序列，设计并合成特异性引物，通过RT-PCR扩增获得了水貂IL-4基因序列全长399 bp，编码132个氨基酸，与雪貂氨基酸序列同源性高达99.2%，与熊猫、犬同源性均为90.0%。将编码成熟蛋白基因构建到原核表达载体pProEX-HTb，IPTG诱导后，SDS-PAGE及Western blotting结果显示，IL-4表达产物为15 ku的包涵体蛋白。通过His-Trap HP亲和层析预装柱变性、复性洗脱可获得高纯度的重组IL-4蛋白。本试验为水貂IL-4基因的进一步研究奠定了基础。

关键词: 水貂; 白介素-4; 序列分析; 原核表达

Abstract:

In order to obtain high purity of interleukin 4 (IL-4) protein,the specific primers were designed and synthesized according to the mink IL-4 gene sequence in GenBank. The IL-4 gene was amplified by RT-PCR using total RNA of the lymphocyte isolated from the peripheral blood that induced by phytohemagglutinin (PHA). The length of IL-4 gene sequence was 399 bp which encoded 132 amino acids. Phylogenetic analysis revealed that the amino acid sequences homology between mink and ferret was 99.2%,90.0% with Ailuropoda melanoleuca and Canis familiaris. Prokaryotic expression vector pProEX-HTb-IL-4 was constructed and induced by IPTG. The recombinant protein of 15 ku was isolated by SDS-PAGE and detected by Western blotting. Highly purified recombinant protein of mink IL-4 was obtained by His-Trap HP affinity columns method. This research laid the foundation for the further studies on the biological function of mink IL-4 gene.

Key words: mink; interleukin-4; sequence analysis; prokaryotic expression

中图分类号:

Q785

范思宁, 张海玲, 赵建军, 王洋, 胡博, 吕爽, 马凡舒, 李欣彤, 凌明玉, 赵辉, 廉士珍, 闫喜军. 水貂白细胞介素-4基因的克隆、序列分析与原核表达[J]. 《中国畜牧兽医》, 2017, 44(1): 30-37.

FAN Si-ning, ZHANG Hai-ling, ZHAO Jian-jun, WANG Yang, HU Bo, LV Shuang, MA Fan-shu, LI Xin-tong, LING Ming-yu, ZHAO Hui, LIAN Shi-zhen, YAN Xi-jun. Cloning,Sequence Analysis and Prokaryotic Expression of Mink Interleukin-4 Gene[J]. , 2017, 44(1): 30-37.

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水貂白细胞介素-4基因的克隆、序列分析与原核表达

Cloning,Sequence Analysis and Prokaryotic Expression of Mink Interleukin-4 Gene

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