猪NLRP6蛋白的原核表达、纯化及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2019.09.026

摘要/Abstract

摘要： 试验旨在表达、纯化猪NLRP6蛋白，并制备鼠抗NLRP6多克隆抗体。根据猪NLRP6全基因核苷酸序列（GenBank登录号：XM_003124236.4）设计特异性引物，利用PCR方法从pMD-19T-NLRP6重组载体中扩增NLRP6基因片段，将扩增产物与原核表达载体pET-32a（+）连接，获得重组质粒pET-32a-NLRP6，经抗性筛选阳性菌、双酶切鉴定、PCR及测序分析后，将其转化大肠杆菌Rosetta（DE3）感受态细胞中，并进行IPTG诱导表达及Western blotting鉴定。对获得的重组融合蛋白进行可溶性分析，经变性、镍柱亲和纯化、复性后得到纯化的融合蛋白，将其免疫BALB/c小鼠制备多克隆抗体。结果显示，试验成功克隆大小约为576 bp的NLRP6基因序列，经鉴定重组质粒pET-32a-NLRP6构建正确，通过IPTG诱导获得大小约34 ku的NLRP6重组融合蛋白，Western blotting分析表明其与小鼠抗6×His单克隆抗体呈阳性反应。可溶性分析结果显示，NLRP6重组融合蛋白以包涵体形式存在，约占95%。纯化的NLRP6重组融合蛋白免疫BALB/c小鼠获得多克隆抗体，经Western blotting分析显示出特异性反应。本试验成功制备了具有免疫原性的NLRP6蛋白及其鼠源多克隆抗体，为NLRP6蛋白生物学功能及相关疾病致病机制研究提供了基础材料。

关键词: 猪NLRP6蛋白; 克隆; 原核表达; 多克隆抗体

Abstract: The aim of the experiment was to express and purify porcine NLRP6 protein and prepare mouse anti-NLRP6 polyclonal antibody.Specific primers were designed according to the nucleotide sequence of porcine NLRP6 (GenBank accession No.:XM_003124236.4).NLRP6 gene fragment was amplified from pMD-19T-NLRP6 recombinant vector by PCR method,and the amplified product was connected with prokaryotic expression vector pET-32a(+) to obtain recombinant plasmid pET-32a-NLRP6.After identification,PCR and sequencing analysis,it was transformed into competent cells of Escherichia coli Rosetta(DE3),and expressed by IPTG and identified by Western blotting.The recombinant fusion protein was purified by denaturation,nickel affinity purification and renaturation.The recombinant fusion protein was immunized to BALB/c mice to prepare polyclonal antibodies.The results showed that a 576 bp NLPR gene was successfully cloned,and the recombinant plasmid pET-32a-NLRP6 was constructed correctly.The recombinant fusion protein of NLRP6 with a size of about 34 ku was obtained by IPTG induction.Western blotting analysis showed that the recombinant protein was positive for mouse anti-6×His monoclonal antibody.The solubility analysis showed that the recombinant fusion protein of NLRP6 existed in the form of inclusion bodies,accounting for about 95%.The purified NLRP6 recombinant fusion protein was used to immunize BALB/c mice to obtain polyclonal antibodies.Western blotting analysis showed specific reactions.The immunogenic NLRP6 protein and its mouse-derived polyclonal antibodies were successfully prepared in this experiment,which provided basic materials for the study of biological functions of NLRP6 protein and pathogenesis of related diseases.

Key words: porcine NLRP6 protein; cloning; prokaryotic expression; polyclonal antibody

中图分类号:

S828

杨文静, 李玉鹏, 梁冬梅, 王倩, 杨升, 乔家运, 姚胜宁, 李海花. 猪NLRP6蛋白的原核表达、纯化及其多克隆抗体的制备[J]. 中国畜牧兽医, 2019, 46(9): 2707-2714.

YANG Wenjing, LI Yupeng, LIANG Dongmei, WANG Qian, YANG Sheng, QIAO Jiayun, YAO Shengning, LI Haihua. Prokaryotic Expression,Purification and Polyclonal Antibody Preparation of Porcine NLRP6 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2707-2714.

参考文献

[1] 温哲,唐朝晖.NLRP6的研究进展[J].中华临床医师杂志(电子版),2014,8(15):2847-2851. WEN Z,TANG Z H.Research progress of NLRP6[J].Chinese Journal of Clinicians (Electronic Edition),2014,8(15):2847-2851.(in Chinese)
[2] TING P Y,LOVERING R C,ALNEMRI E S,et al.The NLR gene family:A standard nomenclature[J].Immunity,2008,28(3):2875-287.
[3] KANNEGANTI T D.Central roles of NLRs and inflammasomes in viral infection[J].Nature Reviews Immunology,2010,10(10):688-698
[4] 彭辉,张涌.NLRP蛋白家族的研究进展[J].中国畜牧杂志,2014,50(3):90-93. PENG H,ZHANG Y.Research progress of NLRP protein family[J].Chinese Journal of Animal Science,2014,50(3):90-93.(in Chinese)
[5] 温哲.NLRP6对HCT116细胞增殖和迁移的影响及在大肠癌组织的表达[D].衡阳:南华大学,2015. WEN Z.The influence of NLRP6 on HCT116 cells'proliferation and migration and expression on colorectal cancer[D].Hengyang:University of South China,2015.(in Chinese)
[6] 李天亮,韩超峰,WANG P,等.NLRP6调节肠道抗病毒天然免疫效应[J].中国肿瘤生物治疗杂志,2016,23(1):88. LI T L,HAN C F,WANG P,et al.NLRP6 regulates intestinal antiviral natural immune effects[J].Chinese Journal of Cancer Biotherapy,2016,23(1):88.(in Chinese)
[7] TIAN X,PASCAL G,MONGET P.Evolution and functional divergence of NLRP genes in mammalian reproductive systems[J].BMC Evolutionary Biology,2009,9(1):202.
[8] 张孝忠,刘一凡,谷思颖,等.流行性乙型脑炎病毒NS1基因克隆及其蛋白表达纯化[J].中国畜牧兽医,2018,45(8):2320-2326. ZHANG X Z,LIU Y F,GU S Y,et al.Cloning,expression and purification of NS1 gene of epidemic encephalitis type B[J].China Animal Husbandry & Veterinary Medicine,2018,45(8):2320-2326.(in Chinese)
[9] 李海花.松鼠葡萄球菌脱皮毒素C的细胞毒性作用以及对先天性免疫的影响[A].中国畜牧兽医学会动物传染病学分会.中国畜牧兽医学会家畜传染病学分会第七届全国会员代表大会暨第十三次学术研讨会论文集(下册)[C].2009. LI H H.Cytotoxicity of squirrel staphylococcal cutaneous toxin C and its effect on innate immunity[A].Branch of Animal Infectious Diseases,CAAV.Proceedings of the 7th National Congress and the 13th Academic Symposium of the Animal Husbandry and Veterinary Society of China Animal Husbandry and Veterinary Society (Volume 2)[C].2009.(in Chinese)
[10] 牛健,于晓旭,李雪,等.重组CARDs TX融合蛋白的表达纯化与复性[J].天津医科大学学报,2014,20(3):184-187. NIU J,YU X X,LI X,et al.Expression,purification and renaturation of recombinant CARDs TX fusion protein[J].Journal of Tianjin Medical University,2014,20(3):184-187.(in Chinese)
[11] KAWAI T,AKIRA S.TLR signaling[J].Cell Death and Differentiation,2007,13(1):24-32.
[12] 孟庆才,朱鏐娈,马雅銮,等.炎性小体的研究进展[J].中华实验和临床感染病杂志(电子版),2012,6(4):79-82. MENG Q C,ZHU L L,MA Y L,et al.Advance in research of inflamasome[J].Chinese Journal of Experimental and Clinical Infectious Disease (Electronic Version),2012,6(4):79-82.(in Chinese)
[13] 潘娜,王开,齐宇,等.模式识别受体在肠炎中的功能及信号转导[J].中国兽医杂志,2016,52(3):73-75. PAN N,WANG K,QI Y,et al.Function and signal transduction of pattern recognition receptors in enteritis[J].Chinese Journal of Veterinary Medicine,2016,52(3):73-75.(in Chinese)
[14] 黄萃园,张洪,白瑞丹.Toll样受体4重组质粒及稳转HepG2细胞株的构建[J].安徽医药,2018,22(9):1714-1717. HUANG C Y,ZHANG H,BAI R D.Establishment of HepG2 cell lines stably transfected with TLR4 gene[J].Anhui Medical and Pharmaceutical Journal,2018,22(9):1714-1717.(in Chinese)
[15] 印磊,陈钟.Toll样受体4在肝细胞肝癌中的研究进展[J].世界华人消化杂志,2016,4:535-541. YIN L,CHEN Z.Role of Toll-like receptor 4 in hepatocellular carcinoma[J].World Chinese Journal of Digestology,2016,4:535-541.(in Chinese)
[16] 汪茂荣.抗人TLR4单克隆抗体(mhTLR4抗体)的制备及其生物活性研究[A].全国中医药防治感染病学术大会暨江苏省中西医结合肝病学术年会[C].2011. WANG M R.Preparation of anti-human TLR4 monoclonal antibody (mhTLR4 antibody) and its biological activity study[A].Annual Meeting of Liver Disease Branch of Jiangsu Association of Traditional and Western Medicine,and Annual Meeting of infection Disease Branch of China Association of Chinese Medicine[C].2011.(in Chinese)
[17] 吴双,朱善元,王永娟,等.鸡Toll样受体2的克隆表达及多克隆抗体的制备[J].江苏农业学报,2014,30(3):586-589. WU S,ZHU S Y,WANG Y J,et al.Cloning of chicken Toll-like receptor 2 gene and preparation of its polyclonal antibody[J].Jiangsu Journal of Agricultural Sciences,2014,30(3):586-589.(in Chinese)
[18] LEVY M,SHAPIRO H,THAISS C A,et al.NLRP6:A multifaceted innate immune sensor[J].Trends in Immunology,2017,38(4):248-260.
[19] 吕翼.NLRP3/6在anisomycin抗肝细胞肝癌中的作用及其机制[D].广州:暨南大学,2018. LYU Y.Role and its mechanism of NLRP3/6 in anisomycin-mediated anti-hepatocellular carcinoma[D].Guangzhou:Jinan University,2018.(in Chinese)
[20] KEMPSTER S L,BELTEKI G,FORHEAD A J,et al.Developmental control of the Nlrp6 inflammasome and a substrate,IL-18,in mammalian intestine[J].American Journal of Physiology-Gastrointestinal and Liver Physiology,2010,300(2):G253-63.
[21] ANAND P K,KANNEGANTI T D.NLRP6 in infection and inflammation[J].Microbes and Infection,2013,15(10-11):661-668.
[22] ANAND P K,MALIREDDI R K S,LUKENS J R,et al.NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens[J].Nature,2012,488(7411):389-393.
[23] NORMAND S,DELANOYE-CRESPIN A,BRESSENOT A,et al.Nod-like receptor pyrin domain-containing protein 6(NLRP6) controls epithelial self-renewal and colorectal carcinogenesis upon injury[J].Proceedings of the National Academy of Sciences,2011,108(23):9601-9606.
[24] 王海滨.NLRP6在胃癌组织中的表达水平对胃癌细胞增殖能力影响的研究[D].福州:福建医科大学,2017. WANG H B.The study about the NLRP6 expression level in gastric cancer and the effect of proliferation in gastric cancer cells[D].Fuzhou:Fujian Medical University,2017.(in Chinese)