Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein

doi:10.15933/j.cnki.1004-3268.2022.02.015

Journal of Henan Agricultural Sciences ›› 2022, Vol. 51 ›› Issue (2): 127-132.DOI: 10.15933/j.cnki.1004-3268.2022.02.015

• Animal Science Veterinary Medicine • Previous Articles Next Articles

Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein

LUO Jun^1,2，DANG Lu³，ZHENG Luping^1,2，LIU Jinling^1,2，CHAI Shujun^1,2，ZHAO Dong^1,2，YANG Yanyan^1,2，TENG Man^1,2

（1. Key Laboratory of Animal Immunology，Henan Academy of Agricultural Sciences/Key Laboratory of Animal Immunology，the Ministry of Agriculture and Rural Affairs of China/Henan Provincial Key Laboratory of Animal Immunology，Zhengzhou 450002，China；2.UK‑China Centre of Excellence for Research on Avian Diseases，Henan Academy of Agricultural Sciences，Zhengzhou 450002，China；3.The Third Affiliated Hospital of Guangzhou Medical University，Guangzhou 510095，China）

Received:2021-11-03 Published:2022-02-15 Online:2022-04-18

鸡异质核糖核蛋白AB单克隆抗体的制备及鉴定

罗俊^1,2，党露³，郑鹿平^1,2，刘金玲^1,2，柴书军^1,2，赵东^1,2，杨艳艳^1,2，滕蔓^1,2

（1.河南省农业科学院动物免疫学重点实验室/农业农村部动物免疫学重点实验室/河南省动物免疫学重点实验室，河南郑州 450002；2.河南省农业科学院中英禽病国际研究中心，河南郑州 450002；3.广州医科大学附属第三医院，广东广州 510095）

通讯作者: 滕蔓（1977-），女，河南周口人，副研究员，博士，主要从事家禽免疫抑制病与肿瘤病研究。E-mail：tm135@aliyun.com
作者简介:罗俊（1978-），男，河南罗山人，研究员，博士，主要从事动物病毒学研究。E-mail：luojun593@aliyun.com
基金资助:
国家自然科学基金项目（U21A20260）；河南省重点研发与推广专项（212102110097）；河南省自然科学基金面上项目（212300410359）；河南省农业科学院自主创新项目（2022ZC65）

Abstract

Abstract: In order to generate the specific monoclonal antibody（mAb）against chicken heterogeneous nuclear ribonucleoprotein AB（hnRNPAB） for its future function study，the recombinant prokaryotic expression plasmid pET‑30a‑hnRNPAB was constructed and transformed into E.coli BL21 competent cells for the IPTG‑induced protein expression.The soluble recombinant hnRNPAB protein was purified by Ni affinity chromatography resin and used as antigen to immunize Balb/c mice.Cell fusion technology was performed to develop the hybridomas. One mAb named as 5H5‑H1 against chicken hnRNPAB protein was screened by indirect enzyme‑linked immunosorbent assay （iELISA）.The indirec timmunofluorescence assay（IFA）and Western blot analysis indicated that the 5H5‑H1 mAb specifically reacted to hnRNPAB protein expressed in chicken embryo fibroblast（CEF）.Furthermore，the IFA and indirect immunocytochemistry assay（ICA）demonstrated that in addition to chicken hnRNPAB protein，the mAb 5H5‑H1 also recognized the native hnRNPAB proteins expressed in five commonly usedmammalian cell lines derived from human，hamster，monkey，bovine and swine.

Key words: Chicken, hnRNP, Monoclonal antibody, IFA, Western blot, ICA

摘要： 为研制鸡异质核糖核蛋白AB（hnRNPAB）单克隆抗体，为其生物学功能研究提供关键试剂，构建重组质粒pET-30a-hnRNPAB，转化感受态细胞E.coli BL21，经IPTG诱导表达，并通过树脂Ni对可溶性表达的鸡hnRNPAB 蛋白进行亲和层析纯化之后免疫Balb/c 小鼠，利用细胞融合技术制备抗鸡hnRNPAB的单克隆抗体细胞株，最终通过间接酶联免疫吸附试验（iELISA）筛选获得1 株单克隆抗体杂交瘤细胞株5H2-H1。间接免疫荧光试验（IFA）和蛋白质免疫印迹（Western blot）分析表明，5H5-H1单克隆抗体可特异性识别鸡胚成纤维细胞（CEF）表达的hnRNPAB蛋白。同时IFA和间接免疫细胞化学试验（ICA）结果还显示，除了特异性识别鸡hnRNPAB蛋白之外，5H5-H1单克隆抗体还可以与源自人、鼠、猴、牛和猪在内的5种常见哺乳动物细胞系表达的hnRNPAB发生特异性反应。

关键词: 鸡, 异质核糖核蛋白, 单克隆抗体, 间接免疫荧光试验, Western blot分析, 间接免疫细胞化学试验

CLC Number:

S855.3

LUO Jun, DANG Lu, ZHENG Luping, LIU Jinling, CHAI Shujun, ZHAO Dong, YANG Yanyan, TENG Man. Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein[J]. Journal of Henan Agricultural Sciences, 2022, 51(2): 127-132.

罗俊, 党露, 郑鹿平, 刘金玲, 柴书军, 赵东, 杨艳艳, 滕蔓. 鸡异质核糖核蛋白AB单克隆抗体的制备及鉴定[J]. 河南农业科学, 2022, 51(2): 127-132.

Figures/Tables 6

References

［1］GEUENS T，BOUHY D，TIMMERMAN V，et al.The hnRNP family：Insights into their role in health and disease［J］.Hum Genet，2016，135（8）：851‑867.

［2］HAN S P，TANG Y H，SMITH R.Functional diversity of the hnRNPs：Past，present and perspectives［J］.Biochem J，2010，430（3）：379‑392.

［3］CARPENTER B，MACKAY C，ALNABULSI A，et al.The roles of heterogeneous nuclear ribonucleoproteins in tumour development and progression ［J］.Biochim Biophys Acta，2006，1765（2）：85‑100.

［4］CAO Y，ZHANG W，JIN Y T，et al.Mining the prognostic value of HNRNPAB and its function in breast carcinoma［J］.Int J Genomics，2020，2020：3750673.

［5］AKINDAHUNSI A A，BANDIERA A，MANZINI G.Vertebrate 2xRBD hnRNP proteins：A comparative analysis of genome，mRNA and protein sequences［J］.Comput Biol Chem，2005，29（1）：13‑23.

［6］CZAPLINSKI K，KOCHER T，SCHELDER M，et al.Identification of 40LoVe，a Xenopus hnRNP D family protein involved in localizing a TGF‑beta‑related mRNA during oogenesis［J］.Dev Cell，2005，8（4）：505‑515.

［7］KROLL T T，SWENSON L B，HARTLAND E I，et al.Interactions of 40LoVe within the ribonucleoprotein complex that forms on the localization element of Xenopus Vg1 mRNA［J］. Mech Dev，2009，126（7）：523‑538.

［8］DICHMANN D S，FLETCHER R B，HARLAND R M.Expression cloning in Xenopus identifies RNA‑binding proteins as regulators of embryogenesis and Rbmx as necessary for neural and muscle development［J］.Dev Dyn，2008，237（7）：1755‑1766.

［9］LEIN E S，HAWRYLYCZ M J，AO N，et al.Genome‑wide atlas of gene expression in the adult mouse brain［J］.Nature，2007，445：168‑176.

［10］TAGA Y，MIYOSHI M，OKAJIMA T，et al.Identification of heterogeneous nuclear ribonucleoprotein A/B as a cytoplasmic mRNA‑binding protein in early involution of the mouse mammary gland［J］.Cell Biochem Funct，2010，28（4）：321‑328.

［11］LAMPASONA A A，CZAPLINSKI K. Hnrnpab regulates neural cell motility through transcription of Eps8［J］.

RNA，2019，25（1）：45‑59.

［12］ZHOU Z J，DAI Z，ZHOU S L，et al.HNRNPAB induces epithelial‑mesenchymal transition and promotes metastasis of hepatocellular carcinoma by transcriptionally activating SNAIL［J］.Cancer Res，2014，74（10）：2750‑2762.

［13］DANG L，TENG M，LI H Z，et al.Marek’s disease virus type 1 encoded analog of miR‑155 promotes proliferation of chicken embryo fibroblast and DF‑1 cells by targeting hnRNPAB［J］.Vet Microbiol，2017，207：210‑218.

［14］YANG Y， CHEN Q， PIAO H Y， et al.HNRNPAB‑regulated lncRNA‑ELF209 inhibits the malignancy of hepatocellular carcinoma［J］.Int J Cancer，2020，146（1）：169‑180.

［15］SMIDT M P，RUSSCHEN B，SNIPPE L，et al.Cloning and characterisation of a nuclear，site specific ssDNA binding protein［J］.Nucleic Acids Res，1995，23（13）：2389‑2395.

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Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein

鸡异质核糖核蛋白AB单克隆抗体的制备及鉴定

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