应用RT-PCR技术扩增β-catenin的cDNA序列,并且在基因下游引入6×His标签序列,克隆至表达载体pGEX-4T-1. 经测序鉴定后,将重组质粒转入BL21pLysS感受态细菌,经IPTG诱导表达,采用Glutathione Sepharose 4B和Ni柱分步纯化GST-β-catenin-His融合蛋白. 所得产物经SDS-PAGE检测,在114 kDa处显示特异条带,与GST-β-catenin-His融合蛋白预期分子量相符. 经Western Blotting鉴定,所纯化蛋白能被β-catenin抗体、GST抗体和His抗体识别,为进一步研究β-catenin的功能提供了基础和前提.
The cDNA sequence of β-catenin was amplified through RT-PCR with 6xHis tag added in the gene downstream, cloned into the vector pGEX-4T-1. The plasmid transformed into BL21pLysS was induced by IPTG. The induced fusion protein was purified stepwise using Glutathione Sepharose 4B and Ni columns. The final protein ran on SDS-PAGE with a specific band at the expected size of 114 kDa. Western Blotting showed that the purified protein can be recognized by the anti-β-catenin, anti-GST, and anti-His antibodies, respectively. All these results provide a basis for further study of β-catenin function.
[1] Wang F, Li F, Yang A Q, et al. β-catenin and E-cadherin the expression and significance in ovarian serous tumor[J]. Journal of Nongken Medicine, 2011, 33(1):1-4(in Chinese). 王芳,李锋,杨安强,等. β-catenin和E-cadherin在卵巢浆液性肿瘤中的表达及意义[J]. 农垦医学,2011,33(1):1-4.
[2] Kajiyama H,Shibata K,Ino K,et al.Possible involvement of SDF lalpha/CXCR4-DPPIV axis in TGF-betal-induced enhancement of migratory potential in human peritoneal mesothelial cells[J].Cell Tissue Res,2007,330(2):221-229.
[3] Lin Y C,Wu M Y,Li D R,et al.Prognostic and elinicopadthological features of E-cadherin,α-catenin,β-catenin,γ-catenin and cycIin D1 expression in humanesophageal squamous cell carcinoma World[J]. Gastroenterol,2004,10(22):3235-3239.
[4] Polakis P.Wnt signaling and cancer[J]. Genes Dev,2000,14(15):1837-1851.
[5] Li H L, He Y J, Qiao C, et al. Expression and significance of β-catenin, cyclinD1 in breast carcinoma[J]. Shanxi Medical Journal, 2011, 40(6):558-560(in Chinese). 李洪利,何艳姣,乔超,等. 乳腺癌β-catenin cyclinD1的表达及意义[J]. 山西医药杂志,2011,40(6):558-560.
[6] Fieke E M,Christine F,Claude C,et al. Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-β-catenin signaling to slow tumor progression[J]. Gastroenterology, 2011,141(4):1486-1497.
[7] Andrzej W,Mariusz K,Stanislaw S,et al. Comparison of Beta-catenin with TGF-beta1,HIF-1alpha and patients disease-free survival in Human colorectal cancer[J].Pathology & Oncology Research,2010,16(3):311-318.
[8] Zhao X L, Duester G. Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension[J]. Gene Expression Patterns,2009,9(6):430-435.
[9] Lindsay J G, Rob B. Redistribution of β-catenin in response to EGF and lithium signalling in human oesophageal squamous carcinoma cell lines[J]. Cancer Cell International,2003,3(1):13.
