Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody

Abstract

Abstract: Objective To prepare the recombinant murine Toll-like receptor-2 N-terminal(mTLR-2N) fusion protein and obtain anti-mTLR-2N polyclonal antibody.Methods The gene encoding 153 amino acids of mTLR-2N was amplified by PCR and cloned into pET32A vector with sequence verification.The recombinant fusion protein was expressed in E.coli and purified by Probond resin column.Rabbits were immunized with fusion protein to obtain the polyclonal anti-sera,and the antibodies were identified by enzyme-linked immunosorbent assay(ELISA) and immunohistochemistry.Results The recombinant fusion protein was efficiently expressed and purified.The polyclonal antibodies could bind to the fusion protein expressed in different vectors as the antigens in ELISA,and also bind with RAW264.7 cells expressing mTLR-2 and CHO cells transfected with full-length mTLR-2 gene.Conclusion The recombinant mTLR-2N fusion protein is obtained and the anti-mTLR-2N polyclonal antibody can recognize natural mTLR-2 on the cell surface.

CLC Number:

R392

YANG Cui-lan1,ZHAO Wen-zhong2,LIU Yan-jun1,ZHU Ping1,FU Ning1 1Department of Immunology,Southern Medical University,Guangzhou 510515,China;2College of Basic Medicine,Sun Yat-sen University,Guangzhou 510080,China. Cloning and expression of murine Toll-like receptor-2 N terminal and preparation of its antibody[J]. Journal of Southern Medical University, 2006, 26(11): 1609-.

References

Related Articles 15

[1]	CHEN Geng-zhen, HU Hui, XU Ming-yan, DENG Xiao-ling . Construction of recombinant adenovirus containing TK gene and its effect against human liver cancer cells [J]. Journal of Southern Medical University, 2010, 30(08): 1887-1889.
[2]	LI De-liang,MA Wen-li,SHI Yong-xia,LI Ling,ZHANG Bao,ZHENG Wen-ling Institute of Genetic Engineering,Southern Medical University,Guangzhou 510515,China. Construction of a eukaryotic expression plasmid containing gp120 gene of HIV-1 subtype B and its expression in HepG2 cells [J]. Journal of Southern Medical University, 2006, 26(12): 1724-1727.
[3]	GUO Yan1,WANG Ju-fang2,LUO Xiao-chun2,SU Jin3,MA Li3,ZHANG Yang-de1 1Xiangya Medical School of Central South University,Changsha 410078,China;2School of Biosciences and Bioengineering,South China University of Technology,Guangzhou 510640,China;3Institute of Molecular Immunology,Southern Medical University,Guangzhou 510515,China. Construction of a recombinant HBV DNA vaccine and its evaluation for specific cellular immune response in mice [J]. Journal of Southern Medical University, 2006, 26(12): 1803-1805.
[4]	XIAO Xin-li1,HAN Jun2,ZHANG Lin3,CHEN Lan4,ZHANG Jin5,CHEN Xin-lin1,ZHOU Wei2,JIANG Hui-ying2,ZHANG Bao-yun2,LIU Yong1,DONG Xiao-ping21Institue of Neurobiology,School of Medicine,Xi’an Jiaotong University,Xi’an 710061,China;2National Institute for Viral Disease Control and Prevention. Protein expression of human neuron-specific enolase and its antiserum preparation [J]. Journal of Southern Medical University, 2006, 26(11): 1543-1547.
[5]	HU Zhi-ming1, MA Li1, ZHOU Ming-qian1, GAO Ji-min1, WANG Xiao-ning1, 2 1Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, China; 2School of Biosciences & Bioengineering, South China University of Technology, Guangzhou 510641, China. Refolding and purification of recombinant human VEGF-121 expressed as inclusion bodies in Escherichia coli [J]. Journal of Southern Medical University, 2006, 26(08): 1083-1086.
[6]	WEN Qian1, MA Li1, SU Jin1, LUO Wei1, WANG Xiao-Ning2 1Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China; 2School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China. Expression, purification and identification of mouse granulocyte-macrophage colony- stimulating factor [J]. Journal of Southern Medical University, 2006, 26(08): 1124-1127.
[7]	MA Shu-dong1, LUO Rong-cheng1, DING Zhen-hua2, LU Feng3, YUAN Chang-qing2 1Department of Oncology, 3Department of Plastic Surgery, Nanfang Hospital, 2Department of Radiation Medicine, Institute of Tropical Medicine, Southern Medical University, Guangzhou 501515, China. Changes in immune function of dendritic cells infected by recombinant adenovirus containing Her2/neu gene of extracellular and transmembrane domain proteins [J]. Journal of Southern Medical University, 2006, 26(08): 1184-1187.
[8]	LI Yan, BAO Lang, ZHANG Hui-dong, LI Ya-sha, ZHU Hai-long Laboratory of Infection and Immunity, College of Preclinical and Forensic Medicine, West China Medical Center of Sichuan University, Chengdu 610041, China. Construction of recombinant Mycobacterium smegmatis expressing ESAT-6 and its effects on macrophages [J]. Journal of Southern Medical University, 2006, 26(07): 923-926.
[9]	ZOU Hong-Yun1, MA Li1, YAO Xin-sheng1, WEN Qian1, LUO Wei1, WANG Xiao-Ning2 1Institute of Molecular Immunology, School of Biotechnology, Southern Medical University, Guangzhou 510515, China; 2School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510641, China. Effects of T cell receptor gene rearrangement on BV CDR3 in Jurkat cells [J]. Journal of Southern Medical University, 2006, 26(07): 939-943.
[10]	LIU Bei-yi, ZHU Ping, LUO Hai-bo, FU Ning Department of Immunology, Southern Medical University, Guangzhou 510515, China. Screening of short peptides binding to cell surface interleukin-2 receptor α chain [J]. Journal of Southern Medical University, 2006, 26(07): 971-974.
[11]	HU Dong-mei1, CAO Yong-qing2, CHEN Zhu-qin3, WANG Ni2 1Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China; 2State Key Laboratory of Reproductive Biology, Beijing 100080, China; 3Department of Obstetrics and Gynecology, Daping Hospital, Third Military Medical University, Chongqing 400042, China. Effect of exogenous cytokine-stimulated decidual cells of early pregnancy on IgG secretion of B lymphocytes [J]. Journal of Southern Medical University, 2006, 26(07): 1050-1052.
[12]	GUO Jie-biao1, GUO Rui2, LIU Yan-hua3 1Zodolabs Biotechnology Limited Company, Nanchang 330029, China; 2Prevention and Treatment Center for Occupational Diseases of Shaoguan, Shaoguan 512026, China; 3Medical College of Shaoguan University, Shaoguan 512026, China. Measurement of affinity constant of monoclonal antibody by competitive antibody binding to antigen [J]. Journal of Southern Medical University, 2006, 26(07): 1057-1059.
[13]	ZHANG Ming-xia, ZHOU Fu-yuan, DIAO Zhi-hong, HE Hai-tang, HOU Jin-Lin LUO Kang-xian Department of Infectious Diseases, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. Stable expression of HBV C gene mutants in immortalized human B-cell lines [J]. Journal of Southern Medical University, 2006, 26(06): 725-729.
[14]	WANG San-bin1, GUO Kun-yuan2, HU Deng-ming1, YIN Bo1 1Department of Hematology, Kunming General Hospital of Chengdu Command, Kunming 650032, China; 2Department of Hematology, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China. Role of TJU103 in prevention of graft-versus-host disease after allogeneic stem cell transplantation in mice [J]. Journal of Southern Medical University, 2006, 26(06): 810-813.
[15]	HUA Jia-ye1,HUANG Ren-wei2 1Second Affiliated Hospital of Guangzhou Medical College,Guangzhou 510260,China;2Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China. In vitro study of cytotoxic T lymphocyte activation by antigen-loaded dendritic cells for killing of K562 cells [J]. Journal of Southern Medical University, 2006, 26(05): 617-619.