GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

Chemical Research in Chinese Universities ›› 2009, Vol. 25 ›› Issue (4): 500-505.

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GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

LI Jiang^1*, YANG Nan-yang², GUAN Xin-gang², ZHANG Shu-zhi², ZHANG Yan¹, QIN Mei-ling², MA Tong-hui² and LI Xiao-meng^2*

1. Dental Hospital, Jilin University, Changchun 130041, P. R. China;
2. Membrane Channel Research Laboratory, School of Life Sciences, Northeast Normal University, Changchun 130024, P. R. China

Received:2008-06-17 Revised:2008-08-25 Online:2009-07-25 Published:2009-10-16
Contact: LI Jiang. E-mail: lijiang69@yahoo.com.cn; LI Xiao-meng. E-mail: lixm441@nenu.edu.cn
Supported by:
Supported by the National Natural Science Foundation of China(Nos.30700827 and 30871301), Jilin Provincial Science & Technology Department of China(Nos.20070719 and 20080731) and Northeast Normal University, China(Nos.20070401, NENUSTC07005).

Abstract

Abstract:

Aquaporins(AQPs) are specific membrane channels for water and other small nonionic molecules. In order to overcome the difficulties to generate the effictive antibody of membrane protein, we selected the cytoplasmic C-terminus of Aquaporin 1(AQP1) as an unique antigen. The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System. On the basis of the resonable amounts of soluable membrane protein peptides, we prepared the specific antibody. To pursure this object, we constructed pGEX-4T-1/mAQP1(DNA sequence from 700 to 801 bp) recombinant plasmid and transformed it into Escherichia coli BL21 cells. The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein. Then we immunized the New Zealand rabbits to prepare the antiserum. The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control. Finally, we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control. We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody, which was consistent with previous report. The successful design and preparation of AQP1 antibody through GST technique is an example as ma- king antibodies against a specific membrane protein.

Key words: Aquaporin 1; GST fusion protein; Polyclonal antibody; Gene knockout mice; Membrane protein

LI Jiang*, YANG Nan-yang, GUAN Xin-gang, ZHANG Shu-zhi, ZHANG Yan,....... GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1[J]. Chemical Research in Chinese Universities, 2009, 25(4): 500-505.

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GST Fusion Protein Based Specific Polyclonal Antibody Preparation of Mouse Aquaporin 1

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