鸡ELAVL1基因克隆、原核表达及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.05.008

Abstract

Abstract: 【Objective】 This study was aimed to clone,express and bioinformatics analyze the CDS region of chicken embryonic lethal abnormal vision like 1 (ELAVL1) gene.【Method】 The CDS region of ELAVL1 gene was amplified by PCR using the cDNA of chicken embryonic fibroblast (CEF) as a template to construct pMD19-T-ELAVL1 clone plasmid.The pET-32a-ELAVL1 prokaryotic recombinant expression plasmid was constructed using pMD19-T-ELAVL1 template.IPTG was used to induce the expression of the recombinant strain,and the recombinant protein expression were detected by SDS-PAGE and Western blotting. Bioinformatics analysis of ELAVL1 protein was performed by online software. 【Results】 The length of ELAVL1 gene CDS in chicken was 981 bp and encoded 326 amino acids, which had the closet relationship with duck and the furthest relationship with zebrafish.SDS-PAGE and Western blotting results showed that the recombinant protein was mainly expressed in soluble form,and its molecular weight was about 55 ku.Bioinformatics analysis showed that the molecular weight of ELAVL1 protein was 36.10 ku,which was a non-fat soluble,weakly alkaline hydrophilic stable protein.The ELAVL1 protein contained 1 N-glycosylation site,3 O-glycosylation sites,32 phosphorylation sites and 10 linear B cell epitope binding site.Subcellular localization revealed that the protein was located in the nucleus.The secondary and tertiary structures were mainly composed of alpha helix and random coil. The ELAVL1 protein contained 3 RNA recognition motif (RRM), which were predicted to interact with various RNA-binding proteins and protein kinases.【Conclusion】 This study successfully cloned the CDS region of ELAVL1 gene in chicken,and ELAVL1 was a non-fat soluble and weakly basic hydrophilic stable protein.After induced expression,soluble expression of ELAVL1 recombinant protein was successfully obtained.The results provided scientific basis for further function of chicken ELAVL1 gene.

Key words: chicken; ELAVL1 gene; clone; bioinformatics analysis

CLC Number:

LIU Jialong, ZHANG Yiwen, ZHANG Yueran, SUN Qijuan, CHEN Jian, HE Lei, JIA Yanyan, DING Ke, YU Zuhua. Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1807-1817.

References

[1] YUTARO U,TOMOKI C,RYOTA K,et al.Post-transcriptional regulation of inflammation by RNA binding proteins via cis-elements of mRNAs[J].Journal of Biochemistry,2019,166(5):375-382.
[2] 曹楠婧,王九菊.RNA结合蛋白HuR的研究进展[J].生命的化学,2021,41(6):1222-1229. CAO N J,WANG J J.Research progress of RNA binding protein HuR[J].Chemistry of Life,2021,41(6):1222-1229.(in Chinese)
[3] DOLLER A,PFEILSCHIFTER J,EBERHARDT W.Signalling pathways regulating nucleo-cytoplasmic shuttling of the mRNA-binding protein HuR[J].Cellular Signalling,2008,20(12):2165-2173.
[4] 陈禹鑫,殷文婕,蒋壮,等.RNA结合蛋白ELAVL1与肿瘤进展的关系研究[J].中国现代医学杂志,2021,31(13):65-70. CHEN Y X,YIN W J,JIANG Z,et al.Review on relationship between RNA-binding protein ELAVL1 and tumor progression[J].China Journal of Modern Medicine,2021,31(13):65-70.(in Chinese)
[5] GRAMMATIKAKIS I,ABDELMOHSEN K,GOROSPE M.Posttranslational control of HuR function[J].Wiley Interdisciplinary Reviews-RNA,2017,8(1):1-15.
[6] KIM H,ABDELMOHSEN K,LAL A,et al.Nuclear retention of HuR through phosphorylation by Cdk1[J].Genes&Development,2008,22(13):1804-1815.
[7] SAUNUS J,FRENCH J,EDWARDS S,et al.Posttranscriptional regulation of the breast cancer susceptibility gene BRCA1 by the RNA binding protein HuR[J].Cancer Research,2008,68(22):9469-9478.
[8] KANG M,RYU B,LEE M,et al.NF-kappaB activates transcription of the RNA-binding factor HuB,via PI3K-AKT signalg,to promote gastric tumorigenesis[J].Gastroenterlogy,2008,135(6):2030-2042.
[9] YU X,LI Y,DING Y,et al.HuR promotes ovarian cancer cell proliferation by regulating TIMM44 mRNA stability[J].Cell Biochemistry and Biophysics,2020,78(4):447-453.
[10] YOUNG L,SANDUJA S,BEMIS-STANDOLI K,et al.The mRNA binding proteins HuR and tristetraprolin regulate cyclooxygenase 2 expression during colon carcinogenesis[J].Gastroenterology,2009,136(5):1669-1679.
[11] JEHUNG J,KITAMURA T,MATSUDA Y,et al.Adenovirus infection induces HuR relocalization to facilitate virus replication[J].Biochemical and Biophysical Research Communications,2018,495(2):1795-1800.
[12] BONENFANT G,WILLIAMS N,NETZBAND R,et al.Zika virus subverts stress granules to promote and restrict viral gene expression[J].Journal of Virology,2019,93(12):1-22.
[13] MUNDT E,VAKHARIA V.Synthetic transcripts of double-stranded Birnavirus genome are infectious[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(20):11131-11136.
[14] CAMPOS A R,GROSSMAN D,WHITE K.Mutant alleles at the locus elav in Drosophila melanogaster lead to nervous system defects.A developmental-genetic analysis[J].Journal of Neurogenetics,1985,2(3):197-218.
[15] MA W,CHENG S,CAMPBELL C,et al.Cloning and characterization of HuR,a ubiquitously expressed Elav-like protein[J].The Journal of Biological Chemistry,1996,271(14):8144-8151.
[16] 张凯琳,汪书华,石松,等.HuR基因克隆及其在肝癌细胞中的表达[J].现代肿瘤医学,2018,26(1):1-4. ZHANG K L,WANG S H,SHI S,et al.HuR gene clone and construction of its eukaryotic expressional vector[J].Journal of Modern Oncology,2018,26(1):1-4.(in Chinese)
[17] 李涛,于文燕,欧小利,等.带FLAG标签小鼠HuR真核表达载体的构建及其生物学功能的鉴定[J].解放军医学杂志,2011,36(4):352-355. LI T,YU W Y,OU X L,et al.Construction of a eukaryotic expression plasmid pcDNA3.1-HuR-FLAG and its transient expression in NIH3T3 cells[J].Medical Journal of Chinese People's Liberation Army,2011,36(4):352-355.(in Chinese)
[18] WAKAMATSU Y,WESTON J.Sequential expression and role of Hu RNA-binding proteins during neurogenesis[J].Development,1997,124(17):3449-3460.
[19] 陈静,吴燕,李梅,等.丝状支原体山羊亚种LPPA蛋白原核表达及间接ELISA方法的建立[J].中国畜牧兽医,2022,49(1):318-327. CHEN J,WU Y,LI M,et al.Prokaryotic expression of LPPA protein of Mycoplasma mycoides subsp.capri and establishment of indirect ELISA method[J].China Animal Husbandry&Veterinary Medicine,2022,49(1):318-327.(in Chinese)
[20] FAN X,STEITZ J.HNS,a nuclear-cytoplasmic shuttling sequence in HuR[J].Proceedings of the National Academy of Sciences of the United States of America,1998,95(26):15293-15298.
[21] OKANO H,DARNELL R.A hierarchy of Hu RNA binding proteins in developing and adult neurons[J].Journal of Neuroscience,1997,17(9):3024-3037.
[22] 营瑞文,张永生,夏欢,等.驴Zfy基因cDNA克隆及生物信息学分析[J].中国畜牧兽医,2018,45(1):39-46. YING R W,ZHANG Y S,XIA H,et al.cDNA cloning and bioinformatics analysis of Zfy gene in Equus asinus[J].China Animal Husbandry&Veterinary Medicine,2018,45(1):39-46.(in Chinese)
[23] COHEN P.The role of protein phosphorylation in human health and disease.[J].European Journal of Biochemistry,2001,268(19):5001-5010.
[24] AHMED S,KABIR M,ARIF M,et al.DeepPPSite:A deep learning-based model for analysis and prediction of phosphorylation sites using efficient sequence information[J].Analytical Biochemistry,2021,612(16):113955.
[25] LAFARGA V,CUADRADO A,LOPEZ DE SILANES I,et al.p38 Mitogen-activated protein kinase-and HuR-dependent stabilization of p21(Cip1) mRNA mediates the G (1)/S checkpoint[J].Molecular and Cellular Biology,2009,29(16):4341-4351.
[26] BASSAGANAS S,CARVALHO S,DIAS A,et al.Pancreatic cancer cell glycosylation regulates cell adhesion and invasion through the modulation of α2β1 integrin and E-cadherin function[J].PLoS One,2014,9(5):e98595.
[27] KOTTA-LOIZOU I,GIAGINIS C,THEOCHARIS S.Clinical significance of HuR expression in human malignancy[J].Medical Oncology,2014,31(9):161-179.
[28] SIDI T,KEASAR C.Redundancy-weighting the PDB for detailed secondary structure prediction using deep-learning models[J].Bioinformatics,2020,36(12):3733-3738.
[29] HINMAN M,LOU H.Diverse molecular functions of Hu proteins[J].Cellular and Molecular Life Sciences,2008,65(20):3168-3181.

Cloning,Prokaryotic Expression and Bioinformatics Analysis of ELAVL1 Gene in Chicken

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	DU Yuanjun, DUAN Guochao, LIU Wei, LI Dapeng, HAN Haixia, LEI Qiuxia, ZHOU Yan, LIU Jie, WANG Jie, CAO Dingguo, LI Fuwei, CHEN Fu. Effect of Translucent on Egg Quality and Its Relationship with Intestinal Function of Laying Hen [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2305-2311.
[2]	WANG Haorui, YU Baojun, YANG Jiayi, WEI Rong, FU Xi, WANG Chuanchuan, ZHANG Di, LI Desheng, CAI Zhengyun, GU Yaling, ZHANG Juan. Bioinformatics Analysis and Tissue Expression of DERA Gene in Jingyuan Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1764-1773.
[3]	WU Jingxuan, SUN Zheng, MA Xuelian, ZHANG Liyang, LIAO Xiudong, LUO Xugang, LI Sufen, LYU Lin. A Survey on Magnesium Contents in Feed Resources for Livestock and Poultry in China [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1888-1898.
[4]	ZHUO Na, ZHU Zhongwu, LI Jing, HU Shixiang, WANG Jianchang, LIN Hua, CHEN Chaolin, HAN Diangang, AI Jun, CHEN Peifu. Bioinformatics Analysis and Eukaryotic Expression of ORF2 Protein of Swine Hepatitis E Virus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1959-1970.
[5]	JIANG Shanyi, ZHOU Bijun, WEN Ming, WANG Kaigong. Prevention of Traditional Chinese Medicine with Probiotics on Eimeria tenella [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2092-2102.
[6]	LIU Xin, CHEN Qun, QIU Yulang, LI Lin, HOU Guoxi, YAN Xiaogang, LI Zhonghe, CHEN Long, GAO Xingai, LIU Dongmei, ZHAI Di, YAN Qiuliang. Research Progress on the Production Mechanism and Control Technology of Chicken Manure Composting Nitrogen-containing and Sulfur-containing Waste Gases [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 2166-2174.
[7]	FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.
[8]	WU Zhi, ZHANG Cong, YUAN Huisha, DONG Hongyan, DAI Lixia, JIA Luqi, WU Shuang, ZHU Shanyuan. Isolation,Identification,Virulence and Drug Resistance of Salmonella from Yellow Chicken [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1695-1706.
[9]	JIA Xinyue, MA Jing, ZHANG Yanyan, MA Xun, WANG Zhengrong, BO Xinwen. Bioinformatics Analysis and Prokaryotic Expression of EGR-07243 and EGR-07243 Genes of Echinococcus granulosus [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 847-858.
[10]	HUANG Xiaoqian, YUAN Junzhao, LIU Jinhe, ZOU Hui, GU Jianhong, YUAN Yan, LIU Xuezhong, LIU Zongping, BIAN Jianchun. Protective Effect of Honokiol on Cadmium-induced Renal Injury in Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1007-1015.
[11]	WEI Yirong, ZHENG Zihua, ZHANG Sanbao, SONG Ying, JIANG Haiyu, SUN Wenyue, CHENG Pengfei, LIU Yufan, ZOU Jianwei, HUANG Yanna, PAN Yan, JIANG Qinyang. Cloning,Bioinformatics Analysis and Tissue Expression of UCP1 Gene in Nubian Goats [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 440-450.
[12]	LYU Shuangfei, MA Xun, WANG Jing, KONG Cuilian, KOU Lijun, LIU Caixia, SHI Weidi, KANG Lichao, REN Huijie. Prokaryotic Expression and Immunoreactivity of PA-LF1 Fusion Protein of Bacillus anthracis [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 674-683.
[13]	LI Xinran, ZHANG Qingyan, ZHA Weiwei, ZHU Miao. Bioinformatics Analysis and Expression of Wnt Gene Family in Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 15-25.
[14]	XIE Jingjing, REN Mengting, FAN Jiahui, JIAO Yaru, HAN Zongxi, MA Deying. Study on Immune Response of Infectious Bronchitis Virus to Chickens and Antiviral Effect of Recombinant Avian Beta-defensins 11 and 12 [J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 246-259.
[15]	ZHANG Hao, LIU Yixuan, ZHU Huiyuan, ZHANG Pengwei, LEI Yanru, GAO Chaoqun, LI Donghua, KANG Xiangtao. Bioinformatics Analysis of Differential Genes of Skin Feather Follicles in Chickens [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3273-3282.