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Polyclonal Antibody Preparation and Application of C.reinhardtii LZTFL1 Protein |
LIU Yan-Xia, FAN Zhen-Chuan |
Institute of Health Biotechnology, International Collaborative Research Center for Health Biotechnology, College of Food Science and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China |
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Abstract Leucine zipper transcription factor 1 (LZTFL1) is a protein associated with cilia signaling, and it is unclear about its function and mechanism in signal transduction. While Chlamydomonas reinhardtii as a model organism that studies the signal transmission of cilia, there is little research on the LZTFL1 protein, and no corresponding antibody has been detected. Based on this phenomenon, polyclonal antibody of LZTFL1 was prepared for subsequent study. The aim was to clone and express C. reinhardtii Lztfl1 gene in Escherichia coli (E. coli). The Lztfl1 gene of C.reinhardtii CC125 was amplified by RT-PCR, then was sub-cloned and heterologous expressed in E.coli BL21(DE3) using pET-28a (+) expression vector. Fusion protein 6×His-LZTFL1 was purified and immunized into New Zealand white rabbit so as to prepare polyclonal antibody. Finally, the sensitivity and specificity of the polyclonal antibody were detected through indirect ELISA and Western blot, respectively. The anti-LZTFL1 showed high sensitivity (1:512 000) and specificity. The above results indicate that Lztfl1 gene has been successfully cloned and expressed, and the rabbit anti-C. reinhardtii LZTFL1 polyclonal antibody has been prepared, which laid the foundation for the study of the structure and function of LZTFL1 protein in C. reinhardtii and its interaction in cilia signal transduction.
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Received: 08 May 2017
Published: 15 November 2017
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[1] |
Merchant Sabeeha S, Prochnik Simon E, Vallon O, et al. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science, 2007, 318(5848):245-250.
|
|
|
[2] |
Badano J L, Mitsuma N, Beales P L, et al. The ciliopathies:an emerging class of human disorders. Annu Rev Genomics Hum Genet, 2006, 7:125-148.
|
|
|
[3] |
Aldahmesh M A, Li Y, Alhashem A, et al. IFT27, encoding a small GTPase component of IFT particles, is mutated in a consanguineous family with Bardet-Biedl syndrome. Hum Mol Genet, 2014, 23(12):3307-3315.
|
|
|
[4] |
Schaefer E, Lauer J, Durand M, et al. Mesoaxial polydactyly is a major feature in Bardet-Biedl syndrome patients with LZTFL1(BBS17) mutations. Clin Genet, 2014, 85(5):476-481.
|
|
|
[5] |
Forsythe E, Beales P L. Bardet-Biedl syndrome. Eur J Hum Genet, 2013, 21(1):8-13.
|
|
|
[6] |
Marion V, Stutzmann F, Gerard M, et al. Exome sequencing identifies mutations in LZTFL1, a BBSome and smoothened trafficking regulator, in a family with Bardet——Biedl syndrome with situs inversus and insertional polydactyly. J Med Genet, 2012, 49(5):317-321.
|
|
|
[7] |
Marion V, Mockel A, De Melo C, et al. BBS-induced ciliary defect enhances adipogenesis, causing paradoxical higher-insulin sensitivity, glucose usage, and decreased inflammatory response. Cell Metab, 2012, 16(3):363-377.
|
|
|
[8] |
Zhang Q, Nishimura D, Seo S, et al. Bardet-Biedl syndrome 3(Bbs3) knockout mouse model reveals common BBS-associated phenotypes and Bbs3 unique phenotypes. Proc Natl Acad Sci U S A, 2011, 108(51):20678-20683.
|
|
|
[9] |
Yen H J, Tayeh M K, Mullins R F, et al. Bardet-Biedl syndrome genes are important in retrograde intracellular trafficking and Kupffer's vesicle cilia function. Hum Mol Genet, 2006, 15(5):667-677.
|
|
|
[10] |
Mykytyn K, Mullins R F, Andrews M, et al. Bardet-Biedl syndrome type 4(BBS4)-null mice implicate Bbs4 in flagella formation but not global cilia assembly. Proc Natl Acad Sci USA, 2004, 101(23):8664-8669.
|
|
|
[11] |
Kim J C, Badano J L, Sibold S, et al. The Bardet-Biedl protein BBS4 targets cargo to the pericentriolar region and is required for microtubule anchoring and cell cycle progression. Nat Genet, 2004, 36(5):462-470.
|
|
|
[12] |
Jiang J, Promchan K, Jiang H, et al. Depletion of BBS protein LZTFL1 affects growth and causes retinal degeneration in mice. J Genet Genomics, 2016, 43(6):381-391.
|
|
|
[13] |
Kiss H, Kedra D, Kiss C, et al. The LZTFL1 gene is a part of a transcriptional map covering 250 kb within the common eliminated region 1(C3CER1) in 3p21.3. Genomics, 2001, 73(1):10-19.
|
|
|
[14] |
Eguether T, San Agustin J T, Keady B T, et al. IFT27 links the BBSome to IFT for maintenance of the ciliary signaling compartment. Dev Cell, 2014, 31(3):279-290.
|
|
|
[15] |
Seo S, Zhang Q, Bugge K, et al. A novel protein LZTFL1 regulates ciliary trafficking of the BBSome and Smoothened. PLoS Genet, 2011, 7(11):e1002358.
|
|
|
[16] |
Wang Z, Fan Z C, Williamson S M, et al. Intraflagellar transport (IFT) protein IFT25 is a phosphoprotein component of IFT complex B and physically interacts with IFT27 in Chlamydomonas. PLoS One, 2009, 4(5):e5384.
|
|
|
[17] |
Fan Z C, Bird R C. An alternative -1/+2 open reading frame exists within viral N(pro)(1-19) region of bovine viral diarrhea virus SD-1. Virus Res, 2012, 163(1):341-351.
|
|
|
[18] |
董彬, 吴淞, 王晶, 等. 莱茵衣藻纤毛内运送蛋白IFT27的原核表达纯化及多克隆抗体制备. 生物技术学报, 2016, 26(6):532-538. Dong B, Wu S, Wang J, et al. Prokaryotic expression,purification and polyclonal antibody preparation of the Chlamydomonas Reinhardtii intraflagellar transport protein (IFT27). Biotechnology, 2016, 26(6):532-538.
|
|
|
[19] |
任海月,董彬,樊振川,等. 莱茵衣藻纤毛内运输蛋白IFT46的原核表达纯化及其多克隆抗体的制备. 生物工程学报, 2016, 32(8):1124-1132. Ren H Y, Dong B, Fan Z C, et al. Prokaryotic expression and purification of Chlamydomonas reinhardtii intraflagellar transport protein 46(IFT46) and preparation of polyclonal antibody. Chinese Journal of Biotechnology, 2016, 32(8):1124-1132.
|
|
|
[20] |
Valcourt U, Gouttenoire J, Aubert-Foucher E, et al. Alternative splicing of type Ⅱ procollagen pre-mRNA in chondrocytes is oppositely regulated by BMP-2 and TGF-beta1. FEBS Lett, 2003, 545(2-3):115-119.
|
|
|
[21] |
田伟,董彬,李振芳,等. 莱茵衣藻IFT139蛋白抗原的原核表达、纯化及多克隆抗体的制备.天津科技大学学报, 2016, 31(8):27-33. Tian W, Dona B, Li Z F, et, al. Prokaryotic expression,purification and polyclonal antibody preparation of Chlamydomonas reinhardtii IFT139 protein antigen. Journal of Tianjin University of Science & Technology, 2016, 31(8):27-33.
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