鸡Visfatin蛋白的原核表达、纯化及其活性研究

doi:10.11843/j.issn.0366-6964.2016.09.006

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (9): 1785-1794.doi: 10.11843/j.issn.0366-6964.2016.09.006

鸡Visfatin蛋白的原核表达、纯化及其活性研究

张盼盼^#，商鹏飞^#，田方圆，韩瑞丽，蒋瑞瑞，孙桂荣，康相涛，刘小军，田亚东^*

（河南农业大学牧医工程学院，河南省家禽种质资源创新工程研究中心，郑州 450002）

收稿日期:2016-03-02 出版日期:2016-09-23 发布日期:2016-09-23
通讯作者: 田亚东，副教授，博士，研究生导师，主要从事家禽营养和家禽生产研究，E-mail：ydtian111@163.com
作者简介:张盼盼(1989-)，女，河南商丘人，硕士，主要从事家禽分子营养研究，E-mail：jqzxzpp2013@126.com；商鹏飞（1986-），女，河南济源人，硕士，主要从事家禽分子营养研究，E-mail：shangpengfei13@163.com。二者同为第一作者
基金资助:
国家自然科学基金(31372330)；教育部创新团队发展计划(IRT1236)；农业部农业科研杰出人才及其创新团队；河南省重大科技专项（151100110800）

Prokaryotic Expression，Purification and Bioactivity Identification of Recombinant Chicken Visfatin Protein

ZHANG Pan-pan ^#，SHANG Peng-fei ^# ，TIAN Fang-yuan，HAN Rui-li，JIANG Rui-rui，SUN Gui-rong，KANG Xiang-tao，LIU Xiao-jun，TIAN Ya-dong^*

(Henan Innovative Engineering Researching Center of Poultry Germplasm Resources，College of Animal Science and Veterinary Medicine，Henan Agricultural University，Zhengzhou 450002，China)

Received:2016-03-02 Online:2016-09-23 Published:2016-09-23

摘要/Abstract

摘要：

本试验旨在通过克隆Visfatin基因，将其在原核表达系统中表达并纯化，通过3T3-L1细胞的分化验证其活性，成功获得鸡重组Visfatin蛋白。以AA肉仔鸡肝组织总RNA为模板，qRT-PCR扩增Visfatin基因，转化进入pGEM-T载体，再与原核表达载体pET-30a连接转化，获取含pET-30a-Visfatin重组质粒的E.coli BL21(DE3)表达菌株。以IPTG诱导重组菌株，优化表达条件，SDS-PAGE鉴定表达情况。采用镍离子亲和层析对Visfatin蛋白进行纯化，Western blot鉴定表达蛋白，并通过3T3-L1细胞的分化情况鉴定表达蛋白Visfatin的活性。结果，通过NcoⅠ、XhoⅠ酶切获得的结果与预期目的条带大小相符，成功构建pET-30a-Visfatin表达载体。SDS-PAGE检测结果表明，在培养基pH为8.0，IPTG终浓度为0.4 mmol•L^-1，30 ℃条件下,诱导10～12 h时可溶性蛋白表达量最大。通过镍离子层析纯化Visfatin蛋白，在SDS-PAGE的60 ku处可见一条与预期一致的蛋白条带。Western blot证明纯化蛋白可与6*His标签特异性结合。油红O染色结果表明，与对照组相比Visfatin诱导的3T3-L1细胞有更多的脂滴形成。qRT-PCR结果显示，在3T3-L1细胞分化过程中，Visfatin作用下标志基因PPARγ、aP2、FAS 和C/EBPα的表达量显著增加（P<0.05）。本研究成功建立了一套标准化的鸡Visfatin重组蛋白表达程序，获得了具有生物活性的鸡Visfatin，为其在家禽领域的进一步研究奠定了基础。

Abstract:

The objective of the present study was to obtain recombinant chicken Visfatin protein with biological activity.The total RNA was extracted from livers of AA broilers.The Visfatin cDNA was first amplified by qRT-PCR and cloned into pGEM-T vector.The cDNA was then cut out from pGEM-T vector by digestion with endonuclease NcoⅠ and XhoⅠand inserted into corresponding cloning sites of the plasmid pET-30a.The positive recombinant plasmid designated as pET-30a-Visfatin was transformed into E.coli BL21 (DE3) competent cells.The cells containing pET-30a-Visfatin were identified.The conditions of IPTG induction were optimized by examining the expression level of recombinant protein using SDS-PAGE analysis.The recombinant protein was purified by Nickel ion affinity chromatography.The identification of the purified recombinant protein was checked by Western blot and the bioactivity was examined by looking into differentiation status of 3T3-L1 cells.A fragment which was the same size to prediction was observed when the recombinant plasmid pET-30a-Visfatin was digested with endonuclease NcoⅠ and XhoⅠ.It indicated that the recombinant expression vector was constructed successfully.The SDS-PAGE analysis revealed that the optimum conditions of induction were 0.4 mmol•L^-1 IPTG，pH 8.0，at 30 ℃ for 10-12 h.The molecular weight of the recombinant protein was about 60 ku，which was the same to prediction.The Western blot analysis indicated that the recombinant protein could be recognized by anti-His antibody.Additionally，the Oil Red O stain assay showed that there were more lipid droplets formation in 3T3-L1 cells induced by Visfatin than that in the controls.Meanwhile，the results of qRT-PCR revealed that the expression levels of genes involved in cell differentiation such as PPARγ， aP2，FAS and C/EBPα increased significantly (P<0.05) when 3T3-L1 cells induced by Visfatin.This study successfully established a standardized expression program for recombinant chicken Visfatin protein production，and obtained the purified recombinant protein with biological activity，which laid a foundation for further research in the field of poultry.

中图分类号:

S831.2

张盼盼，商鹏飞，田方圆，韩瑞丽，蒋瑞瑞，孙桂荣，康相涛，刘小军，田亚东. 鸡Visfatin蛋白的原核表达、纯化及其活性研究[J]. 畜牧兽医学报, 2016, 47(9): 1785-1794.

ZHANG Pan-pan，SHANG Peng-fei，TIAN Fang-yuan，HAN Rui-li，JIANG Rui-rui，SUN Gui-rong，KANG Xiang-tao，LIU Xiao-jun，TIAN Ya-dong. Prokaryotic Expression，Purification and Bioactivity Identification of Recombinant Chicken Visfatin Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1785-1794.

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鸡Visfatin蛋白的原核表达、纯化及其活性研究

Prokaryotic Expression，Purification and Bioactivity Identification of Recombinant Chicken Visfatin Protein

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