猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备

doi:10.11843/j.issn.0366-6964.2017.03.005

畜牧兽医学报 ›› 2017, Vol. 48 ›› Issue (3): 425-435.doi: 10.11843/j.issn.0366-6964.2017.03.005

猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备

王瑾¹，唐青海^1,2*，谷雅静¹，李晓楠¹，李羽¹，王雪雨¹，姚伦广¹，阚云超^1*

（1. 南阳师范学院河南省伏牛山昆虫生物学重点实验室/昆虫生物反应器河南省工程实验室，南阳 473061；2. 衡阳师范学院生命科学与环境学院生物药物研究所，衡阳 421008）

收稿日期:2016-10-17 出版日期:2017-03-23 发布日期:2017-03-22
通讯作者: 阚云超，教授，硕导，主要从事生物制药与疫苗工程研究，E-mail：kanyunchao@163.com；唐青海，博士，硕导，主要从事病毒分子生物学研究，E-mail：qinghaitang109@126.com
作者简介:王瑾(1982-)，女，蒙古族，河南南阳人，硕士生，主要从事生物制药与疫苗工程研究，E-mail：115431539@qq.com
基金资助:
国家自然科学基金青年基金（31101837）；河南省重点科技攻关项目（142102110101）；衡阳师范学院引进人才专项项目；河南省高等学校重点科研项目（16A230023）

Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody

WANG Jin¹，TANG Qing-hai^1,2* ，GU Ya-jing¹，LI Xiao-nan¹，LI Yu¹，WANG Xue-yu¹，YAO Lun-guang¹，KAN Yun-chao^1*

(1.Henan Provincial Engineering Laboratory of Insect Bio-reactor/Henan Provincial Key Laboratory of Funiu Mountain in Insect Biology, Nanyang Normal University, Nanyang 473061， China; 2. Institute of Biological Medicine, College of Life Sciences and Environment, Hengyang Normal University, Hengyang 421008, China)

Received:2016-10-17 Online:2017-03-23 Published:2017-03-22

摘要/Abstract

摘要：

本研究旨在克隆猪转录因子ELF4基因，体外表达ELF4蛋白并制备抗该蛋白的多克隆抗体。采用RT-PCR扩增猪ELF4基因，运用生物信息学软件分析其核苷酸和编码氨基酸序列特征，将其克隆至原核表达载体pET28a，构建重组表达载体pET28a-pELF4，转化Rosetta（DE3）菌株，在不同温度、IPTG浓度、诱导时间等条件下进行诱导表达，采用SDS-PAGE和Western blot分析蛋白表达，重组pELF4蛋白（rpELF4）经纯化免疫鸡制备抗pELF4 抗体。结果表明，pELF4开放阅读框为1 989 bp （GenBank No.：KU097322），编码662个氨基酸，该基因与已经测定的人、牛、小鼠的ELF4的核苷酸相似性依次为89.4%、90.7%和81.1%，与牛等大动物的亲缘关系相对较近，与人和小鼠亲缘关系相对较远；结果显示，成功表达了重组蛋白pELF4，rpELF4分子量为95 ku，该蛋白以可溶性和包涵体两种形式存在，在37 ℃条件下，以终浓度为0.8 mmol•L^-1 IPTG诱导8 h包涵体表达量达到最高。第4次免疫后第13天，鸡抗rpELF4抗体滴度达到1︰256 000倍以上。本试验利用原核表达系统成功制备了重组pELF4蛋白，并制备了免疫活性和特异性良好的鸡抗pELF4多克隆抗体，为该蛋白生物学功能及相关疾病研究提供了基础材料。

关键词: 猪ELF4蛋白, 原核表达, 免疫

Abstract:

This research aimed to clone and express the porcine transcription factor E74-like factor 4 (pELF4) gene in vitro and to prepare its polyclonal antibody. The pELF4 gene was cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and the characteristics of its nucleotides and coding amino acids were analyzed by the bioinformatics softwares. The recombinant expression vector pET28a-pELF4 was constructed by inserting the pELF4 gene into the prokaryotic expression vector pET28a. Then pET28a-pELF4 was transformed into E. coli Rosetta(DE3) and to investigate its expression under different temperatures, IPTG concentrations and induction time. The expressed proteins were analyzed by SDS-PAGE and Western blot. The anti-pELF4 immuno-globulin egg yolk antibody(IgY) was prepared by immunizing hens after purification of the recombination pELF4 protein (rpELF4). The results showed that the ORF (Open reading frame) of pELF4 gene (GenBank Accession number: KU097322) was 1 989 bp, coding 662 amino acids. The similarity between pELF4 gene and that of human, cattle and mouse were 89.4%, 90.7% and 81.1%, respectively, therefore it was closer to the animals such as cattle while it was further to human and mouse. The relative molecular weight of rpELF4 was 95 ku, and it existed in the form of protein and inclusion body. The amount of inclusion body reached highest when the expression combination was 37℃, 0.8 mmol•L^-1 of IPTG and 8 h.The chicken anti-rpELF4 antibody titer was above 1︰256 000 fold at the 13rd days post the 4th vaccination. The recombinant protein pELF4 was successfully expressed by prokaryotic expression system, the chicken anti-pELF4 polyclonal antibodies with the wonderful activity and specifity was prepared, which provided basic material for the research of the biofunction of this protein and the related diseases.

Key words: porcine ELF4 protein, prokaryotic expression, immunization

中图分类号:

S828
S813.1

王瑾，唐青海，谷雅静，李晓楠，李羽，王雪雨，姚伦广，阚云超. 猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备[J]. 畜牧兽医学报, 2017, 48(3): 425-435.

WANG Jin，TANG Qing-hai，GU Ya-jing，LI Xiao-nan，LI Yu，WANG Xue-yu，YAO Lun-guang，KAN Yun-chao. Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(3): 425-435.

参考文献

［1］SZABO A, RAJNAVOLGYI E. Finding a fairy in the forest: ELF4, a novel and critical element of type I interferon responses ［J］. Cell Mol Immunol, 2014, 11(3): 218-220.
［2］SUICO M A, YOSHIDA H, SEKI Y, et al. Myeloid Elf-1-like factor, an ETS transcription factor, up-regulates lysozyme transcription in epithelial cells through interaction with promyelocytic leukemia protein ［J］. J Biol Chem, 2004, 279(18): 19091-19098.
［3］LU Z, KIM K A, SUICE M A, et al. MEF up-regulates human β-defensin 2 expression in epithelial cells ［J］. FEBS Lett, 2004, 561(1-3):117-121.
［4］SEKI Y, SUICO M A, UTO A, et al. The ETS transcription factor MEF is a candidate tumor suppressor gene on the X chromosome ［J］. Cancer Res, 2002, 62(22): 6579-6586.
［5］SASHIDA G, LIU Y, ELF S, et al. ELF4/MEF activates MDM2 expression and blocks oncogene-induced p16 activation to promote transformation ［J］. Mol Cell Biol, 2009, 29(13): 3687-3699.
［6］SASHIDA G, BAE N, DI GIANDOMENICO S, et al. The Mef/Elf4 transcription factor fine tunes the DNA damage response ［J］. Cancer Res, 2011, 71 (14): 4857-4865.
［7］YAMADA T, PARK C S, MAMONKIN M, et al. Transcription factor ELF4 controls the proliferation and homing of CD8+ T cells via the Krüppel-like factors KLF4 and KLF2 ［J］. Nat Immunol, 2009, 10 (6): 618-626.
［8］YOU F P, WANG P H, YANG L, et al. ELF4 is critical for induction of type I interferon and the host antiviral response ［J］. Nat Immunol, 2013, 14(12): 1237-1246.
［9］LACORAZZA H D, MIYAZAKI Y, DI CRISTOFANO A, et al. The ETS protein MEF plays a critical role in perforin gene expression and the development of natural killer and NK-T cells ［J］. Immunity, 2002, 17(4): 437-449.
［10］张辉, 唐青海, 黄立平, 等. 猪肾细胞源三种蛋白质对猪圆环病毒2型复制的调节作用［J］. 畜牧兽医学报, 2015,46(11): 2040-2049.
ZHANG H, TANG Q H, HUANG L P, et al. Regulation effect of three proteins derived from porcine kidney cells on porcine circovirus type 2 replication［J］. Acta Veterinaria et Zootechnica Sinica, 2015, 46(11): 2040-2049.(in Chinese)
［11］黎帮勇, 胡尚连, 曹颖, 等. 毛竹NAC转录因子家族生物信息学分析［J］. 基因组学与应用生物学, 2015,34(8):1769-1777.
LI B Y, HU S L,CAO Y,et al. Bioinformatics analysis of NAC gene family in Moso bamboo［J］. Genomics and Applied Biology, 2015,34(8): 1769-1777. (in Chinese)
［12］丁蓉, 胡毓华, 何思思, 等. 转录因子THAP1在人肺泡Ⅱ型上皮细胞中结合靶基因的谱系分析［J］. 南京医科大学学报:自然科学版, 2015,35(9): 1200-1204,1215.
DING R, HU Y H, HE S S, et al. Profiling analysis of target genes binded by transcription factor THAP1 in human alveolar typeⅡepithelial cells［J］. Acta Universitatis Medicinalis Nanjing: Natural Science，2015,35(9）：1200-1204,1215. (in Chinese)
［13］许玲, 卫培培, 张大勇, 等. 大豆转录因子基因GmMYB111的克隆及功能分析［J］. 中国农业科学, 2015,48(15): 3079-3089.
XU L, WEI P P, ZHANG D Y,et al. Expression and Function Analysis of the Transcription Factor GmMYB111 in Soybean ［J］. Scientia Agricultura Sinica, 2015,48(15):3079-3089. (in Chinese)
［14］SHI Y L, WANG D, ZHU X Y, et al. Molecular cloning and functional characterization of porcine E74-like factor 4 (ELF4) ［J］. Dev Comp Immunol, 2016, (65): 149-158.
［15］潘滨, 吴建祥, 李桂新, 等. 烟草曲茎病毒复制相关蛋白基因原核表达条件优［J］. 浙江大学学报：农业与生命科学版, 2007, 33(1): 24-28.
PAN B, WU J X, LI G X,et al. Optimization of prokaryotic expression of Tobacco curly shoot virus replication-associated protein gene ［J］.Journal of Zhejiang University: Agriculture & Life Science, 2007,33(1):24-28. (in Chinese)
［16］WEICKERT M J, DOHERTY D H, BEST E A, et al. Optimization of heterologous protein production in Escherichia coli ［J］. Curr Opin Biotechnol, 1996, 7(5): 494-499.
［17］LIU X H, LU W J, DU C H, et al. Studies on fermentation conditions of genetically engineered Escherichial coli expressing the major house dust mite allergen Der p 1 ［J］. Academic Journal of Guangzhou Medical College, 2002, 30(1): 46-48.
［18］王劼, 蒋思文,彭健. 重组K88黏附素亚单位蛋白在大肠杆菌中的诱导表达［J］. 农业生物技术学报, 2006, 14(6): 957-962.
WANG J, JIANG S W, PENG J. Optimization of expression of recombinant K88 fimbrial subunit protein in Escherichia coli ［J］.Journal of Agricultural Biotechnology, 2006, 14(6): 957-962. (in Chinese)
［19］LU H, WU W, ZENG Q Y, et al. The expression analysis of recombinant protein in Escherichia coli BL21 (DE3) ［J］. Journal of Beijing Forestry University, 2001,23(6): 1-4.
［20］BERTRAND F, DEVILLE S B, AROUS J B, 等. Montanide^TMISA 71 VG:具有细胞免疫反应的禽类疫苗用的新佐剂［C］.北京：中国畜牧兽医学会禽病学分会第十六次学术研讨会, 2012.
BERTRAND F, DEVILLE S B, AROUS J B, et al. Montanide^TMISA 71 VG:a novel adjuvant for cellular immune -response in poultry vaccines［C］. Beijing: China Institute of Animal Husbandry and Veterinary Virology, Proceedings of the Sixteenth Symposium，2012. (in Chinese)
［21］JANG S I, LILLEHOJ H S, LEE S H, et al. Montanide^TM ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella ［J］. Exp Parasitol, 2011, 127(1): 178-183.
［22］王彦妮, 曾显营, 田国彬, 等. AA肉鸡免疫禽流感油乳剂灭活疫苗细胞免疫应答的变化［J］. 中国预防兽医学报, 2011, 33(2): 141-145.
WANG Y N, ZENG X Y, TIAN G B,et al. The dynamic changes of cellular immune response of AA broiler immunized with inactivated oil-emulsion vaccines against avian influenza ［J］. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(2): 141-145. (in Chinese)
［23］BENTES G A, LANZARINI N M, LIMA L R P, et al. Using immunoglobulin Y as an alternative antibody for the detection of hepatitis A virus in frozen liver sections ［J］. Mem Inst Oswaldo Cruz, 2015: 110(4): 577-579.
［24］REDDY P, RAMLAL S, SRIPATHY M H, et al. Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference ［J］. J Immunol Methods, 2014, 408: 114-122.
［25］YANG Y E, WEN J L, ZHAO S Q, et al. Prophylaxis and therapy of pandemic H1N1 virus infection using egg yolk antibody ［J］. J Virol Methods, 2014, 206: 19-26.
［26］FENG Y F, LIU W X, SHI D F. Effectiveness of egg yolk antibody against shiga toxin II variant toxicity in vitro and in vivo ［J］. Curr Microbiol, 2013, 67(4): 448-453.
［27］VEGA C, BOK M, SAIF L, et al. Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves ［J］. Res Vet Sci, 2015, 103: 1-10.
［28］ZHANG Q, LIN H, SUI J X, et al. Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot ［J］. J Sci Food Agric, 2015, 95 (1): 136-140.

[1]	韩小红, 何翃闳, 王靖雷, 张慧珠, 马悦, 马进彪, 赵生贤, 余四九, 崔燕, 樊江峰. 牦牛p38MAPK在雌性主要生殖器官中的表达[J]. 畜牧兽医学报, 2019, 50(9): 1802-1812.
[2]	付绍祖, 李露露, 张敬, 李丹, 郑海学. 猪源DDX56对口蹄疫病毒的复制及其对病毒诱导的RLR通路调节的研究[J]. 畜牧兽医学报, 2019, 50(9): 1849-1856.
[3]	高俊莹, 张东超, 李璇, 王华琳, 姜宁. 弓形虫表面抗原SAG1黏附宿主细胞表面硫化肝素的特性研究[J]. 畜牧兽医学报, 2019, 50(9): 1874-1881.
[4]	彭孝坤, 张宇, 黄晓瑜, 周广琛, 邢晓南, 张恩平. 急性冷应激对绵羊免疫功能和不同组织热休克蛋白70家族基因表达的影响[J]. 畜牧兽医学报, 2019, 50(8): 1625-1634.
[5]	彭孝坤, 张宇, 黄晓瑜, 周广琛, 邢晓南, 张恩平. 急性冷应激对绵羊免疫功能和不同组织热休克蛋白70家族基因表达的影响[J]. 畜牧兽医学报, 2019, 50(8): 1625-1634.
[6]	彭孝坤, 赵天, 黄晓瑜, 张宇, 邢晓南, 张恩平. 急性热应激对山羊血液生化指标及血淋巴细胞热休克蛋白70家族基因表达的影响[J]. 畜牧兽医学报, 2019, 50(6): 1219-1229.
[7]	唐泽群, 罗海燕, 葛铭, 郭荣, 许丹蕾, 张瑞莉. 抗chTLR3单克隆抗体制备及其初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1292-1300.
[8]	彭孝坤, 赵天, 黄晓瑜, 张宇, 邢晓南, 张恩平. 急性热应激对山羊血液生化指标及血淋巴细胞热休克蛋白70家族基因表达的影响[J]. 畜牧兽医学报, 2019, 50(6): 1219-1229.
[9]	唐泽群, 罗海燕, 葛铭, 郭荣, 许丹蕾, 张瑞莉. 抗chTLR3单克隆抗体制备及其初步应用[J]. 畜牧兽医学报, 2019, 50(6): 1292-1300.
[10]	倪爱心, 麻慧, 陈继兰. 寄生虫来源的外泌体研究进展[J]. 畜牧兽医学报, 2019, 50(5): 909-917.
[11]	武乐祎, 吴素芳, 车影, 张强, 李鹏, 卞广林, 王选年. 稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析[J]. 畜牧兽医学报, 2019, 50(5): 1056-1063.
[12]	倪爱心, 麻慧, 陈继兰. 寄生虫来源的外泌体研究进展[J]. 畜牧兽医学报, 2019, 50(5): 909-917.
[13]	武乐祎, 吴素芳, 车影, 张强, 李鹏, 卞广林, 王选年. 稳定表达猪圆环病毒2型Cap蛋白的CHO-K1细胞系的建立及免疫原性分析[J]. 畜牧兽医学报, 2019, 50(5): 1056-1063.
[14]	刘同君, 赵盼, 赵敏孟, 刘龙, 崔恒宓, 龚道清, 耿拓宇. 内质网应激标记基因Grp78参与鹅肥肝免疫/炎症状态的调控[J]. 畜牧兽医学报, 2019, 50(4): 727-737.
[15]	韩兴发, 李俊丽, 曹晓涵, 储明星, 曾宪垠. SD雄鼠3种不同新蛋白构型GnRH去势抗原免疫学及生物学效果对比研究[J]. 畜牧兽医学报, 2019, 50(4): 738-749.

猪ELF4基因的克隆、蛋白表达及其多克隆抗体制备

Cloning and Expression of Porcine ELF4 Gene and Preparation of Its Polyclonal Antibody

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价