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人同源异形盒基因HOXB9多克隆抗体的制备和活性鉴定
Preparation and characterization of homeobox B9（HOXB9） polyclonal antibody
目的 制备兔抗人同源异形盒基因HOXB9的多克隆抗体，为后续深入研究HOXB9蛋白的功能提供工具。方法 采用聚合酶链反应(PCR)和 DNA重组技术，构建HOXB9基因5,端7-458碱基片段到原核表达载体pGEX-4T-1和pMal-C2x中，热休克法转化及异丙基-β-D-硫代吡喃半乳糖苷诱导表达谷胱甘肽转移酶标签的HOXB9 N端融合蛋白和麦芽糖结合蛋白标签的HOXB9 N端融合蛋白。纯化GST-HOXB9 N端融合蛋白免疫新西兰白兔，ELISA鉴定效价后，颈动脉放血，收集血清，纯化抗体。免疫印迹法及免疫沉淀法鉴定抗体有效性及特异性。结果 本实验制备的抗体能够特异识别目标分子的单一条带，并且具有较强的免疫沉淀天然构象的HOXB9蛋白的能力；采用所制备的抗体检测发现，HOXB9蛋白在小鼠免疫器官、胰脏、结肠、肺、小脑、雌性生殖系统及睾丸中表达较高，其他器官表达较低或无表达。结论 成功制备了特异而有效的兔抗人HOXB9蛋白多克隆抗体，此多克隆抗体可用于免疫印迹法分析及免疫沉淀实验。
Objective The purpose of this study was to prepare rabbit anti-homeobox B9(HOXB9) polyclonal antibody and to provide a tool for future study. Methods DNA fragments of human HOXB9 gene 5,terminal 7-458 base pairs was respectively cloned into PGEX-4T-1 vector expressing GST tag and pMal-C2x vector expressing MBP tag by polymerasechain reaction and recombinant DNA technology.The GST-HOXB9-N-terminal fusion protein and MBP-HOXB9-N-terminal fusion protein were expressed by E.coli BL21（DE3）after heat shock transformation and IPTG induction. The New Zealand rabbit was immunized with the purified GST-HOXB9-N-terminal fusion protein. We used ELISA assay to determine the titer of the antiserum.If the antiserum was effective, the rabbit blood was obtained from the carotid artery and immunoglobulinG(IgG)was purified from serum. Effectiveness and specificity of the antibody were identified by Western blotting and immunoprecipitation. Results The prepared antibody specifically recognized a single band of the target molecule, and had a strong capability of immunoprecipitation on the native HOXB9 protein.Using this antibody, we demonstrated that HOXB9 expressed highly in the mouse immune organs,pancreas,colon, lung, cerebellum, female reproductive system and testis,while expressed lowly in other organs. Conclusion We successfully prepared a rabbit anti-human HOXB9 polyclonal antibody. This polyclonal antibody is specific and effective, and may be used for Western blotting and immunoprecipitation analysis.
HOXB9 / 纯化蛋白 / 多克隆抗体 / 免疫印迹法 / 新西兰白兔
Homeobox B9 / Fusion protein / Polyclonal antibody / Western blotting / New Zealand rabbit
［1］ Malicki J, Bogarad LD, Martin MM, et al. Functional analysis of the mouse homeobox gene HoxB9 in Drosophila development［J］. Mech Dev,1993,42(3):139-150.
［2］Heffer A, Xiang J, Pick L. Variation and constraint in Hox gene evolution［J］. Proc Natl Acad Sci USA, 2013, 110(6):2211-2216.
［3］Nguyen DX, Chiang AC, Zhang XH, et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis［J］. Cell, 2009, 138(1):51-62.
［4］Kim JH, Kim YH, Han JH, et al. Silencing of homeobox B9 is associated with down-regulation of CD56 and extrathyroidal extension of tumor in papillary thyroid carcinoma［J］.Hum Pathol, 2012, 43(8):1221-1228.
［5］Shrestha B, Ansari KI, Bhan A, et al. Homeodomain-containing protein HOXB9 regulates expression of growth and angiogenic factors, facilitates tumor growth in vitro and is overexpressed in breast cancer tissue ［J］. FEBS J, 2012, 279(19):3715-3726.
［6］Nagel S,Burek C,Venturini L, et al.Comprehensive analysis of homeobox genes in Hodgkin lymphoma cell lines identifies dysregulated expression of HOXB9 mediated via ERK5 signaling and BMI1 ［J］. Blood, 2007, 109(7):3015-3023.
［7］Gorski DH, Walsh K. The role of homeobox genes in vascular remodeling and angiogenesis［J］. Circ Res, 2000, 87(10):865-872.
［8］Rao MK, Maiti S, Ananthaswamy HN, et al. A highly active homeobox gene promoter regulated by Ets and Sp1 family membersin normal granulosa cells and diverse tumor cell types［J］. J Biolog Chemi, 2002, 277(29):26036-26045.
［9］Prevot D, Voeltzel T, Birot AM, et al. The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interactwith the homeoprotein Hoxb9 and enhance its transcriptional activation ［J］. J Biologi Chemi, 2000, 275(1):147-153.
［10］Hayashida T, Takahashi F, Chiba N, et al. HOXB9, a gene overexpressed in breast cancer, promotes tumorigenicity and lung metastasis［J］. Proc Natl Acad Sci USA, 2010, 107(3):1100-1105.
［11］Ansari KI, Shrestha B, Hussain I, et al. Histone methylase MLL1 and MLL3 coordinate with estrogen receptors in estrogen mediated HOXB9 expression［J］ . Biochemistry, 2011, 50(17):3517-3527.
教育部博士点基金（20100001110046）;国家自然科学基金资助项目;自然科学基金资助项目
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